Factors influencing fruit and vegetable intake among urban Fijians: A qualitative study.

Low fruit and vegetable intake is an important risk factor for micronutrient deficiencies and non-communicable diseases, but many people worldwide, including most Fijians, eat less than the World Health Organization recommended amount. The present qualitative study explores factors that influence fruit and vegetable intake among 57 urban Fijians (50 women, 7 men) of indigenous Fijian (iTaukei) and South Asian (Indian) descent. Eight focus group discussions were held in and around Suva, Fiji's capital and largest urban area, which explored motivation for eating fruit and vegetables, understandings of links to health and disease, availability and sources, determinants of product choice, and preferred ways of preparing and eating fruit and vegetables. Data were analysed using thematic content analysis. Regardless of ethnicity, participants indicated that they enjoyed and valued eating fruit and vegetables, were aware of the health benefits, and had confidence in their cooking skills. In both cultures, fruit and vegetables were essential components of traditional diets. However, increasing preferences for processed and imported foods, and inconsistent availability and affordability of high-quality, low-priced, fresh produce, were identified as important barriers. The findings indicate that efforts to improve fruit and vegetable intake in urban Fijians should target the stability of the domestic fruit and vegetable supply and access.

Developmental iron uptake and axonal transport in the retina of the rat.

We examined differently aged postnatal (P) rats for the distribution and uptake of iron in the eye with the main emphasis on iron uptake in the retina. The concentration of iron in the eye was 48 µg/g in rats aged one postnatal day (P1). Then concentration fell to approximately 12 µg/g at P30 and rose to 35 µg/g at P70. Perls' stain which labels both ferrous and ferric iron was found to exhibit a weak labeling in the retina at P1 contrasted by a robust labeling of macrophages found in the choroid of the retina. In older aged rats, the labeling of cells of the retina was much more intense and confined to cells widespread in the layers of the retina. In both P16 and adult rats injected intravenously with [(59)Fe-(125)I]transferrin, the uptake of (59)Fe, estimated as the volume of distribution, was significantly higher than that of [(125)I]transferrin, and uptake of both compounds was higher than that of simultaneously injected [(131)I]albumin. In the P16 rat, the uptake of (59)Fe expressed as the volume of distribution, V(D), rose linearly reaching approximately 2500 nl at 60 min. In the adult rat, the uptake of (59)Fe was of the same magnitude. Comparing P1 and P16 rats, the uptake of (59)Fe, [(125)I]transferrin and [(131)I]albumin was higher at P1 in both eyeball and retina. Emulsion autoradiography of retinas from P16 and adult rats injected with [(55)Fe]transferrin into the vitreous body showed uptake mainly in photoreceptor cells and retinal ganglion cells. Adult rats injected into the vitreous body with [(59)Fe]transferrin showed anterograde axonal transport from the retina into the optic nerve, optic tract, and superior colliculus. Immunoprecipitates of homogenates of the optic nerve revealed that (59)Fe was precipitable with an antibody raised against ferritin, indicative of detachment of iron from transferrin within the axons of the retinal ganglion cells. The data demonstrate an age-dependent but continuous iron uptake by the retina, and are indicative of anterograde axonal transport of transferrin by retinal ganglion cells.

Iron absorption and hepatic iron uptake are increased in a transferrin receptor 2 (Y245X) mutant mouse model of hemochromatosis type 3.

Hereditary hemochromatosis type 3 is an iron (Fe)-overload disorder caused by mutations in transferrin receptor 2 (TfR2). TfR2 is expressed highly in the liver and regulates Fe metabolism. The aim of this study was to investigate duodenal Fe absorption and hepatic Fe uptake in a TfR2 (Y245X) mutant mouse model of hereditary hemochromatosis type 3. Duodenal Fe absorption and hepatic Fe uptake were measured in vivo by 59Fe-labeled ascorbate in TfR2 mutant mice, wild-type mice, and Fe-loaded wild-type mice (2% dietary carbonyl Fe). Gene expression was measured by real-time RT-PCR. Liver nonheme Fe concentration increased progressively with age in TfR2 mutant mice compared with wild-type mice. Fe absorption (both duodenal Fe uptake and transfer) was increased in TfR2 mutant mice compared with wild-type mice. Likewise, expression of genes participating in duodenal Fe uptake (Dcytb, DMT1) and transfer (ferroportin) were increased in TfR2 mutant mice. Nearly all of the absorbed Fe was taken up rapidly by the liver. Despite hepatic Fe loading, hepcidin expression was decreased in TfR2 mutant mice compared with wild-type mice. Even when compared with Fe-loaded wild-type mice, TfR2 mutant mice had increased Fe absorption, increased duodenal Fe transport gene expression, increased liver Fe uptake, and decreased liver hepcidin expression. In conclusion, despite systemic Fe loading, Fe absorption and liver Fe uptake were increased in TfR2 mutant mice in association with decreased expression of hepcidin. These findings support a model in which TfR2 is a sensor of Fe status and regulates duodenal Fe absorption and liver Fe uptake.

Quasi-periodic X-ray brightness fluctuations in an accreting millisecond pulsar.

The relativistic plasma flows onto neutron stars that are accreting material from stellar companions can be used to probe strong-field gravity as well as the physical conditions in the supra-nuclear-density interiors of neutron stars. Plasma inhomogeneities orbiting a few kilometres above the stars are observable as X-ray brightness fluctuations on the millisecond dynamical timescale of the flows. Two frequencies in the kilohertz range dominate these fluctuations: the twin kilohertz quasi-periodic oscillations (kHz QPOs). Competing models for the origins of these oscillations (based on orbital motions) all predict that they should be related to the stellar spin frequency, but tests have been difficult because the spins were not unambiguously known. Here we report the detection of kHz QPOs from a pulsar whose spin frequency is known. Our measurements establish a clear link between kHz QPOs and stellar spin, but one not predicted by any current model. A new approach to understanding kHz QPOs is now required. We suggest that a resonance between the spin and general relativistic orbital and epicyclic frequencies could provide the observed relation between QPOs and spin.

Restricted transport of anti-transferrin receptor antibody (OX26) through the blood-brain barrier in the rat.

Anti-transferrin receptor IgG2a (OX26) transport into the brain was studied in rats. Uptake of OX26 in brain capillary endothelial cells (BCECs) was > 10-fold higher than isotypic, non-immune IgG2a (Ni-IgG2a) when expressed as % ID/g. Accumulation of OX26 in the brain was higher in 15 postnatal (P)-day-old rats than in P0 and adult (P70) rats. Iron-deficiency did not increase OX26 uptake in P15 rats. Three attempts were made to investigate transport from BCECs further into the brain. (i) Using a brain capillary depletion technique, 6-9% of OX26 was identified in the post-capillary compartment consisting of brain parenchyma minus BCECs. (ii) In cisternal CSF, the volume of distribution of OX26 was higher than for Ni-IgG2a when corrected for plasma concentration. (iii) Immunohistochemical mapping revealed the presence of OX26 almost exclusively in BCECs; extravascular staining was observed only in neurons situated periventricularly. The data support the hypothesis of facilitated uptake of OX26 due to the presence of transferrin receptors at the blood-brain barrier (BBB). However, OX26 accumulation in the post-capillary compartment was too small to justify a conclusion of receptor-mediated transcytosis of OX26 occurring in BCECs. Accumulation of OX26 in the post-capillary component may result from a diphasic transport that involves high-affinity accumulation of OX26 by the BCECs, clearly exceeding that of Ni-IgG2a, followed by a second transport mechanism that releases OX26 non-specifically further into the brain. The periventricular localization suggests that OX26 probably also derives from transport across the blood-CSF barrier.

Mechanisms of iron transport into rat erythroid cells.

Three mechanisms of iron uptake by rat erythroid cells were identified, two with non-transferrin-bound iron (NTBI) and one with transferrin-bound iron (Fe-Tf). Uptake of NTBI occurred by a high affinity mechanism (K(m) approximately 0.1 microM). Activity of the high affinity mechanism was maximal in sucrose solution and of the low affinity mechanism in KCl solution. Both were inhibited by NaCl and by certain ion transport inhibitors, but they differed in their sensitivity to the various inhibitors. Fe-Tf uptake was also of high affinity (K(m) 0.1 microM). All the transport mechanisms show higher activity in reticulocytes than in mature erythrocytes, and all could provide iron for heme synthesis in reticulocytes. The results demonstrate certain conditions which should be followed in order to study high affinity transport of NTBI. These include use of a low packed cell volume in the incubation mixture, low iron concentrations (0.01-1.0 microM), short incubation times (up to 20 min), and low osmolality (approximately 200 mOsm/kg) during incubation with the NTBI and subsequent washing of the cells.

Expression of transferrin mRNA in rat oligodendrocytes is iron-independent and changes with increasing age.

As transferrin in the brain may originate principally from synthesis by three different cell types, i.e. hepatocytes, oligodendrocytes and choroid plexus, this study employed a morphological analysis to specifically address oligodendrocytic expression of transferrin mRNA in young (P17) and adult (P50) rats. In spite of a lowering of the concentration of brain iron by approximately 22% in the young iron deficient rats transferrin mRNA expression in oligodendrocytes was not affected when measured by quantitative densitometry. In adult rats, the baseline transferrin mRNA expression in oligodendrocytes was higher than in the young animals, but did not change in spite of a reduction in brain iron by approximately 19%. Brain iron and transferrin mRNA expression in oligodendrocytes were unaltered in iron overloaded rats when compared to age-matched controls. As transferrin expression was lower in the young rat, when constituents from the blood have a relatively higher concentration in the brain than during adulthood, it seems unlikely that blood-borne factors such as transition metals act as inducers of transferrin gene expression in oligodendrocytes. Instead, the higher but constitutive expression of transferrin mRNA at later ages, when the blood-brain barrier segregates the brain from other body parts, may indicate that molecules released from the brain interior are responsible for regulating transcription of the transferrin gene.

Iron excretion in iron-overloaded rats following the change from an iron-loaded to an iron-deficient diet.

BACKGROUND: Iron stores in the body are thought to be regulated by a mechanism associated with the rate of iron absorption from the diet, with no significant role played by iron excretion. We report the existence of an iron excretory process that results in the loss of significant amounts of liver iron. METHODS AND RESULTS: Rats were fed 3% carbonyl iron for 9 weeks, which resulted in a 20-fold increase in liver non-haem iron. When the rats on this iron-loaded diet were switched to a low iron diet for 2 and 7 days, liver non-haem iron levels fell 30% and 45%, respectively. A similar fall in transferrin-bound plasma iron was also seen. As the liver iron had not redistributed to other body compartments, it was concluded that the iron had been excreted and that the excreted iron represented a loss of 22% and 28% in total body non-haem iron over 2 and 7 days, respectively. Ligation of the common bile duct in iron loaded rats that had been switched to the iron-deficient diet was accompanied by a similar loss of liver iron and also hepatocellular damage. In addition, measurement of enterocyte iron levels showed that only approximately 5% of the total iron excreted was found in these cells. CONCLUSION: Neither bile nor enterocytes play a significant role in iron excretion. The similarity in the degree of fall in transferrin-bound iron levels with a change in diet suggests that iron excretion involves the uptake and excretion of transferrin bound-iron, possibly by goblet cells. The observed hypertrophy of the intestinal mucosa associated with carbonyl iron feeding may facilitate hypersecretion of mucous and the excretion of this iron.

Gastrointestinal function, divalent metal transporter-1 expression and intestinal iron absorption.

Iron absorption involves two carriers, one involved in the uptake of iron across the microvillus membrane of the enterocyte and the other in its transfer to the plasma at the basolateral surface. The uptake phase is thought to involve divalent metal transporter-1 (DMT1) which may move from the cytoplasm to the microvillus membrane under conditions of iron deficiency. To examine this possibility we used fasted animals previously fed an iron-deficient diet and then gavaged with iron. We measured the processes of iron absorption using in vivo gut sacs and correlated the changes observed with the intensity of DMT1 staining and gene expression in the duodenum. Fasting resulted in increased iron absorption, whereas gavage with iron decreased absorption. These changes were due to alterations in the uptake phase of absorption but not the transfer phase. There was also a highly significant correlation between the reduction in iron absorption, microvillus DMT1 staining and messenger ribonucleic acid (mRNA) expression. The loss of DMT1 from the microvillus membrane was not associated with an increase in cytoplasmic staining, suggesting that its loss was due to destruction of the carrier protein. It is concluded that DMT1 functional activity is determined by de novo synthesis and that the latter is regulated post-transcriptionally by enterocyte iron levels.

Cellular distribution of ferric iron, ferritin, transferrin and divalent metal transporter 1 (DMT1) in substantia nigra and basal ganglia of normal and beta2-microglobulin deficient mouse brain.

We examined whether high levels of circulatory iron may cause iron accumulation in the brain. In particular, we focussed on the substantia nigra and basal ganglia as several papers have indicated that iron may accumulate here and cause death of dopaminergic neurons. Normal mice and a mouse model of hereditary haemochromatosis, the beta2-microglobulin (beta2m) knock out [beta2m (-/-)] mouse, which has high levels of circulating iron due to increased iron absorption, were examined. The iron concentration in livers were: 170+/-15 microg/g (mean +/- SD) in controls and 1010+/-50 microg/g in beta2m (-/-) mice (p<0.001), whereas in the brain the respective values were 47 +/-1 microg/g and 53+/-2 microg/g (p<0.02). Hence, the difference between cerebral iron levels of normal and beta2m (-/-) mice was small. Histological examination of the brains revealed an unequivocal distribution of ferric iron, ferritin, transferrin and divalent metal transporter 1 (DMT1), which were indistinguishable when normal and beta2m (-/-) mice were compared. In the substantia nigra and basal ganglia, ferric iron and the iron-binding proteins were present in identical cell types, which mainly comprised oligodendrocytes and microglia. Neurons were lightly labelled with transferrin and DMT1. The virtual lack of an increase in cerebral iron in beta2m (-/-) mice clearly shows that the blood-brain barrier (BBB) is capable of restricting the transport of excess plasma iron into the brain.

Gene expression of divalent metal transporter 1 and transferrin receptor in duodenum of Belgrade rats.

Regulation of iron absorption is thought to be mediated by the amount of iron taken up by duodenal crypt cells via the transferrin receptor (TfR)-transferrin cycle and the activity of the divalent metal transporter (DMT1), although DMT1 cannot be detected morphologically in crypt cells. We investigated the uptake of transferrin-bound iron by duodenal enterocytes in Wistar rats fed different levels of iron and Belgrade (b/b) rats in which iron uptake by the transferrin cycle is defective because of a mutation in DMT1. We showed that DMT1 in our colony of b/b rats contains the G185R mutation, which in enterocytes results in reduced cellular iron content and increased DMT1 gene expression similar to levels in iron deficiency of normal rats. In all groups the uptake of transferrin-bound iron by crypt cells was directly proportional to plasma iron concentration, being highest in iron-loaded Wistar rats and b/b rats. We conclude that the uptake of transferrin-bound iron by developing enterocytes is largely independent of DMT1.

Transferrin receptor activity and localisation in the rat duodenum.

It is not known how the efficiency of intestinal iron absorption is regulated. One hypothesis suggests that an interaction between the transferrin receptor (TfR) and the haemochromatosis protein (HFE) regulates the level of iron loading in crypt cells. The hypothesis goes on to suggest that this determines the amount of transport protein, expressed in villus enterocytes, that is involved in iron absorption. Mice with haploinsufficiency for TfR are iron deficient and this is thought to be caused by reduced iron absorption. This suggests that TfR may play a role in the regulation and/or mechanism of iron absorption. We investigated TfR function and distribution by measuring iron uptake from plasma transferrin and by immunohistochemistry. The uptake of transferrin-bound iron (Tf-Fe2) into mucosal cells subsequently separated along the crypt-villus axis was compared to the presence of TfR, determined by immunohistochemistry using frozen and wax sections. Frozen sections showed TfR staining in crypt and villus epithelial cells. In wax sections TfR was only identified in a supranuclear region commencing in enterocytes at the crypt-villus junction and attaining greatest levels at the villus tip. This indicates that the processing of wax tissue exposes a TfR epitope that otherwise remains undetectable when studied in frozen sections. This appearance in paraffin sections was inversely related to the uptake of Tf-Fe2. Supranuclear TfR was not associated with lysosomes, since there was no difference in the uptake of normal Tf-Fe2 and that of the non-digestible cellobiose Tf-Fe2, and Western blot analysis revealed similar amounts of TfR in crypt and villus cells. Also the uptake of Tf-Fe2 increased linearly with time, albeit less in villus than crypt cells, suggesting that maturation of an efflux system in villus cells is not responsible for this difference. We hypothesise that TfR in the supranuclear region of villus enterocytes may play a role in iron absorption.

Characterization of iron uptake from transferrin by murine endothelial cells.

Iron is required by the brain for normal function, however, the mechanisms by which it crosses the blood-brain barrier (BBB) are poorly understood. The uptake and efflux of transferrin (Tf) and Fe by murine brain-derived (bEND3) and lymph node-derived (m1END1) endothelial cell lines was compared. The effects of iron chelators, metabolic inhibitors and the cellular activators, lipopolysaccharide (LPS) and tumour necrosis factor-alpha (TNF-alpha), on Tf and Fe uptake were investigated. Cells were incubated with 59Fe-125I-Tf; Fe uptake was shown to increase linearly over time for both cell lines, while Tf uptake reached a plateau within 2 h. Both Tf and Fe uptake were saturable. bEND3 cells were shown to have half as many Tf receptors as m1END1 cells, but the mean cycling times of a Tf molecule were the same. Tf and Fe efflux from the cells were measured over time, revealing that after 2 h only 25% of the Tf but 80% of the Fe remained associated with the cells. Of 7 iron chelators, only deferriprone (L1) markedly decreased Tf uptake. However, Fe uptake was reduced by more than 50% by L1, pyridoxal isonicotinoyl hydrazone (PIH) and desferrithiocin (DFT). The cellular activators TNF-alpha or LPS had little effect on Tf turnover, but they accelerated Fe uptake in both endothelial cell types. Phenylarsenoxide (PhAsO) and N-ethyl maleimide (NEM), inhibitors of Tf endocytosis, reduced both Tf and Fe uptake in both cell lines, while bafilomycin A1, an inhibitor of endosomal acidification, reduced Fe uptake but did not affect Tf uptake. The results suggest that Tf and Fe uptake by both bEND3 and m1END1 is via receptor-mediated endocytosis with release of Fe from Tf within the cell and recycling of apo-Tf. On the basis of Tf- and Fe-metabolism both cell lines are similar and therefore well suited for use in in vitro models for Fe transport across the BBB.

Transferrin and transferrin receptor function in brain barrier systems.

1. Iron (Fe) is an essential component of virtually all types of cells and organisms. In plasma and interstitial fluids, Fe is carried by transferrin. Iron-containing transferrin has a high affinity for the transferrin receptor, which is present on all cells with a requirement for Fe. The degree of expression of transferrin receptors on most types of cells is determined by the level of Fe supply and their rate of proliferation. 2. The brain, like other organs, requires Fe for metabolic processes and suffers from disturbed function when a Fe deficiency or excess occurs. Hence, the transport of Fe across brain barrier systems must be regulated. The interaction between transferrin and transferrin receptor appears to serve this function in the blood-brain, blood-CSF, and cellular-plasmalemma barriers. Transferrin is present in blood plasma and brain extracellular fluids, and the transferrin receptor is present on brain capillary endothelial cells, choroid plexus epithelial cells, neurons, and probably also glial cells. 3. The rate of Fe transport from plasma to brain is developmentally regulated, peaking in the first few weeks of postnatal life in the rat, after which it decreases rapidly to low values. Two mechanisms for Fe transport across the blood-brain barrier have been proposed. One is that the Fe-transferrin complex is transported intact across the capillary wall by receptor-mediated transcytosis. In the second, Fe transport is the result of receptor-mediated endocytosis of Fe-transferrin by capillary endothelial cells, followed by release of Fe from transferrin within the cell, recycling of transferrin to the blood, and transport of Fe into the brain. Current evidence indicates that although some transcytosis of transferrin does occur, the amount is quantitatively insufficient to account for the rate of Fe transport, and the majority of Fe transport probably occurs by the second of the above mechanisms. 4. An additional route of Fe and transferrin transport from the blood to the brain is via the blood-CSF barrier and from the CSF into the brain. Iron-containing transferrin is transported through the blood-CSF barrier by a mechanism that appears to be regulated by developmental stage and iron status. The transfer of transferrin from blood to CSF is higher than that of albumin, which may be due to the presence of transferrin receptors on choroid plexus epithelial cells so that transferrin can be transported across the cells by a receptor-mediated process as well as by nonselective mechanisms. 5. Transferrin receptors have been detected in neurons in vivo and in cultured glial cells. Transferrin is present in the brain interstitial fluid, and it is generally assumed that Fe which transverses the blood-brain barrier is rapidly bound by brain transferrin and can then be taken up by receptor-mediated endocytosis in brain cells. The uptake of transferrin-bound Fe by neurons and glial cells is probably regulated by the number of transferrin receptors present on cells, which changes during development and in conditions with an altered iron status. 6. This review focuses on the information available on the functions of transferrin and transferrin receptor with respect to Fe transport across the blood-brain and blood-CSF barriers and the cell membranes of neurons and glial cells.

Localisation of divalent metal transporter 1 (DMT1) to the microvillus membrane of rat duodenal enterocytes in iron deficiency, but to hepatocytes in iron overload.

BACKGROUND: The mechanism of iron absorption by the intestine and its transfer to the main iron storage site, the liver, is poorly understood. Recently an iron carrier was cloned and named DMT1 (divalent metal transporter 1). AIMS: To determine the level of DMT1 gene expression and protein distribution in duodenum and liver. METHODS: A DMT1 cRNA and antibody were produced and used in in situ hybridisation and immunohistochemistry, respectively, in rats in which the iron stores were altered by feeding diets with normal, low, and high iron content. RESULTS: Duodenal DMT1 mRNA was low in crypts and increased at the crypt-villus junction in iron deficient and control rats; it fell in the iron loaded state. Staining for DMT1 protein was not detected in crypts. In villus enterocytes, protein staining was localised to the microvillus membrane in iron deficiency, in the cytoplasm and to a lesser extent in the membrane in controls, and entirely in the cytoplasm of iron loaded animals. Liver DMT1 mRNA was distributed evenly across hepatocytes. DMT1 protein staining was observed on hepatocyte plasma membranes, with highest values in the iron loaded state, lower values in control animals, and none after iron depletion. CONCLUSIONS: Results are consistent with a role for DMT1 in the transmembrane transport of non-transferrin bound iron from the intestinal lumen and from the portal blood.

Iron-independent neuronal expression of transferrin receptor mRNA in the rat.

Neuronal transferrin receptor protein expression is highly upregulated widely in CNS following iron deficiency. Using the medial habenular nucleus as a model of neuronal transferrin receptor mRNA expression, the present study examined 17-day-old rats subjected to variations in dietary iron. Changing the iron availability resulted in alterations in plasma and cerebrospinal fluid (CSF) levels of transferrin and iron. The iron-binding capacity of transferrin in CSF was exceeded in normal and iron-overloaded rats. In spite of a lowering of the concentration of brain iron by approximately 22% in iron-deficient rats, neuronal transferrin receptor mRNA was not affected when measured by quantitative densitometry. Brain iron and neuronal transferrin receptor mRNA expression was unaltered in iron overloaded rats. The absence of a rise in transferrin receptor mRNA during iron deficiency suggests that neuronal transferrin receptor mRNA expression is regulated by another mechanism than the post-transcriptional regulation mechanism, which has been attributed to cells of non-neural tissue.

Characterisation of citrate and iron citrate uptake by cultured rat hepatocytes.

BACKGROUND/AIMS: The endogenous low molecular weight iron chelator, citrate, is considered to be an important contributor to iron transport and the liver the main site of uptake of iron citrate in subjects suffering from diseases of iron overload. Moreover, the citrate-metabolising enzyme, aconitase, is implicated in the regulation of cellular iron metabolism. This study was undertaken to determine the role of citrate and ferric citrate in the uptake of iron by rat hepatocytes. METHODS: Cultured rat hepatocytes were incubated (37 degrees C, 15 min) with 100 microM [14C]-citrate in the presence or absence of 1.0 microM 55Fe. Membrane-bound and intracellular radiolabel were separated by incubation with the general protease, Pronase. RESULTS: Our results suggest that ferric citrate uptake is mediated by a specific citrate binding site which exhibits a higher affinity for citrate in the presence of iron than in its absence. Citrate was internalised by hepatocytes, with at least 70% being oxidised to CO2 within 15 min. Citrate uptake was pH-dependent, did not require the presence of sodium and increased with increasing iron concentration. Metabolic energy, anion channels, the Na+, K+-ATPase and vesicle acidification do not appear to play a role in uptake of ferric citrate, but functional sulphydryl groups may be involved. CONCLUSIONS: The data suggest either that ferric citrate complexes with higher molar ratios of iron to citrate relative to the incubation medium are bound preferentially to the membrane, or that once citrate has delivered its iron to the membrane, the complex dissociates and the components are internalised separately.

Evidence for low molecular weight, non-transferrin-bound iron in rat brain and cerebrospinal fluid.

Transferrin (Tf) donates iron (Fe) to the brain by means of receptor-mediated endocytosis of Tf at the brain barriers. As Tf transport through the brain barriers is restricted, Fe is probably released into the brain extracellular compartment as non-Tf-bound iron (NTBI). To evaluate NTBI in the brain and cerebrospinal fluid (CSF), different aged rats (P15, P20, P56) were injected intravenously with [59Fe-125I]Tf followed by sampling of CSF and brain tissue. Between 80 and 93% of 59Fe in CSF was absorbed with anti-Tf and 1 and 5% with anti-ferritin antibodies. The fraction of 59Fe from CSF passing through a 30,000 molecular weight (MW) cutoff filter was approximately 5% (P15), 10% (P20), and 15% (P56). Measurements of Fe and Tf concentrations in CSF of P20 rats revealed that the Fe-binding capacity of Tf was exceeded. In the supernatants of brain homogenates, between 94 and 99% of 59Fe was absorbed with anti-Tf and anti-ferritin antibodies. The respective fractions of 59Fe in the supernatants passing through the 30 kD cutoff filter were 4% (P15), 2% (P20), and 6% (P56). In brain homogenates mixed before filtering with desferroxamine (DFO) or nitrilotriacetic acid (NTA) which complex loosely protein-bound Fe and non-protein-bound Fe, these 59Fe fractions were 2-fold higher. The results indicate that NTBI is present extracellularly in CSF and probably in brain interstitium.

Expression of the neuronal transferrin receptor is age dependent and susceptible to iron deficiency.

In order to characterize the mechanism by which Iron (Fe) is taken up by neurons, we examined the neuronal expression of transferrin receptor (TR) in rats during development and iron (Fe) deficiency by using immunohistochemistry, in vitro receptor autoradiography and in situ hybridization. In contrast to the continuous expression of TR in brain capillary endothelial cells regardless of the age of the animals studied, the expression of neuronal TR was almost absent at late embryonic and early postnatal ages but increased with increasing age to reach a plateau from postnatal (P) 21 through adulthood as verified by immunohistochemical staining. Reducing the Fe stores potentiated the expression of TR immunoreactivity in neurons of both young and adult rats in several grey matter regions. Increased TR immunoreactivity was also observed in neuronal extensions of neurons of the medial habenular nucleus, reticular neurons of the brainstem, and fibers projecting to the area postrema. TR immunoreactivity was never observed in white matter regions, except for that recorded in brain capillaries. In vitro receptor autoradiography verified the increased capacity for transferrin binding during Fe deficiency. By contrast, TR mRNA expression was not affected by Fe deficiency. These findings demonstrate that the expression of the neuronal TR protein is age dependent and susceptible to Fe deficiency.

Transport mechanisms for iron and other transition metals in rat and rabbit erythroid cells.

1. Earlier studies have shown that Fe2+ transport into erythroid cells is inhibited by several transition metals (Mn2+, Zn2+, Co2+, Ni2+) and that Fe2+ transport can occur by two saturable mechanisms, one of high affinity and the other of low affinity. Also, the transport of Zn2+ and Cd2+ into erythroid cells is stimulated by NaHCO3 and NaSCN. The aim of the present investigation was to determine whether all of these transition metals can be transported by the processes described for Fe2+, Zn2+ and Cd2+ and to determine the properties of the transport processes. 2. Rabbit reticulocytes and mature erythrocytes and reticulocytes from homozygous and heterozygous Belgrade rats were incubated with radiolabelled samples of the metals under conditions known to be optimal for high- and low-affinity Fe2+ transport and for the processes mediated by NaHCO3 and NaSCN. 3. All of the metals were transported by the high- and low-affinity Fe2+ transport processes and could compete with each other for transport. The Km and Vmax values and the effects of incubation temperature and metabolic inhibitors were similar for all the metals. NaHCO3 and NaSCN increased the uptake of Zn2+ and Cd2+ but not the other metals. 4. The uptake of all of the metals by the high-affinity process was much lower in reticulocytes from homozygous Belgrade rats than in those from heterozygous animals, but there was no difference with respect to low-affinity transport. 5. It is concluded that the high- and low-affinity 'iron' transport mechanisms can also transport several other transition metals and should therefore be considered as general transition metal carriers.