Indicators of erythrocyte formation and degradation in rats with either vitamin A or iron deficiency.

Vitamin A deficiency produces anemia and altered iron status. In this study with rats we tested two hypotheses regarding vitamin A deficiency: (1) that it impairs erythropoiesis, leading to an increased red cell turnover, and (2) that it inhibits the glycosylation of transferrin. Erythropoietic activity was assessed indirectly by determining the myeloid:erythroid ratio in bone marrow smears, the number of erythroid colonies in the red pulp of spleen, the blood reticulocyte index, and zinc protoporphyrin and plasma transferrin receptor concentrations. Transferrin glycosylation was assessed by measuring the sialic acid content of transferrin. The effects of vitamin A deficiency were compared with those of iron deficiency. Iron deficiency produced anemia and low iron levels in organs. Vitamin A deficiency produced low levels of plasma and hepatic retinol, and it induced decreased plasma total iron-binding capacity and raised iron levels in tibia and spleen. Short- but not long-term iron deficiency reduced the number of erythroid colonies in spleen; vitamin A deficiency had no influence. Neither iron nor vitamin A deficiency influenced the myeloid:erythroid ratio in bone marrow smears and the blood reticulocyte production. Plasma transferrin receptor and erythrocyte zinc protoporphyrin concentrations were not affected by vitamin A deficiency but increased with iron deficiency. Vitamin A deficiency did not stimulate erythrocyte breakdown, as indicated by unaltered plasma lactate dehydrogenase activity and reduced plasma total bilirubin levels. Both vitamin A and iron deficiencies raised the proportion of multiple sialylated transferrins in plasma. Thus, we have not found evidence that vitamin A deficiency affects erythropoiesis and erythrocyte turnover. The iron accumulation in spleen and bone marrow may be related to reduced iron transport due to inhibition of transferrin synthesis rather than inhibition of transferrin sialylation.

Accumulation and release of iron in polarly and non-polarly cultured trophoblast cells isolated from human term placentas.

We investigated the usefulness of membrane grown human term trophoblast cells in transferrin-mediated iron transfer studies. We showed that diferric transferrin is taken up both at the microvillous and at the basal membrane by means of receptor-mediated endocytosis. Uptake from the microvillous side is predominant. This corresponded with a much higher expression of transferrin receptors at the microvillous membrane as compared to the basal one. Iron appeared to accumulate in the cell. Accumulation was higher when transferrin was supplied at the microvillous side. Transfer of iron could not be assessed because uptake of transferrin by the cells was much less than passive diffusion of transferrin through the cell-free filter. The observation of iron accumulation was unexpected for a transfer epithelium. Could it be that part of the iron taken up by the cells is rapidly released whereas the remaining part accumulates? In this case the rate of iron uptake should be higher than the rate of iron accumulation. This question was assessed with non-polarly cultured trophoblast cells. We showed that like in polar cells iron accumulated in ferritin. A new experimental design enabled us to demonstrate that indeed the rate of transferrin-mediated iron is in excess over iron accumulation. We thus provide evidence for a mechanism that enables rapid transfer of iron across the syncytiotrophoblast cell layer.

Characteristics of recovery from the euthyroid sick syndrome induced by tumor necrosis factor alpha in cancer patients.

Cytokines have been implicated in the pathogenesis of the euthyroid sick syndrome. Isolated limb perfusion (ILP) with recombinant human tumor necrosis factor alpha (rTNF) and melphalan in patients with melanoma or sarcoma is accompanied by high systemic TNF levels. We examined the prolonged effects (7 days) of ILP on thyroid hormone metabolism with respect to induction and recovery of the euthyroid sick syndrome in six cancer patients. After ILP, when the limb is reconnected to the systemic circulation, leakage of residual rTNF resulted in systemic peak levels at 10 minutes postperfusion followed by a parallel increase in plasma interleukin-6 (IL-6) and cortisol, with maximum levels at 4 hours (P < .05). A rapid decrease was observed at 5 minutes for plasma triiodothyronine (T3), reverse T3 (rT3), thyroxine (T4), and thyroxine-binding globulin (TBG) (P < .05), whereas free T4 (FT4) and T3-uptake showed a sharp increase, with peak levels at 5 minutes (P < .05). T3, T4, and TBG levels remained low until 24 hours after ILP In contrast, rT3 increased above pretreatment values to maximum levels at 24 hours (P < .05). Plasma thyrotropin (TSH) showed an initial decrease at 4 hours postperfusion (P < .05) but exceeded pretreatment values from day 1 to day 7 (by +94%+/-43% to +155%+/-66%, P < .05), preceding the recovery of T4 and T3 levels. T3 and rT3 returned to initial values at day 4. T4 and TBG levels recovered at day 2. T4 exceeded basal values at days 5 to 7 (P < .05). It is concluded that ILP with rTNF induces a euthyroid sick syndrome either directly or indirectly through other mediators such as IL-6 or cortisol. The recovery from this euthyroid sick syndrome is, at least in part, TSH-dependent, since the prolonged elevation of TSH values preceded and persisted during the normalization of T3 and the elevation of T4 levels. This biphasic pattern of induction of and recovery from the euthyroid sick syndrome may be a general feature of nonthyroidal disease. The euthyroid sick syndrome should be interpreted not only in relation to the presence of nonthyroidal diseases but also in relation to the recovery from these diseases.

Structure, function and clinical significance of transferrin receptors.

Iron plays an essential role in a spectrum of metabolic processes. Cellular iron uptake is facilitated by transferrin receptor (TfR)-mediated endocytosis. In recent years more insight has been obtained in TfR physiology and the regulation of cellular iron homeostasis. The synthesis of TfR and the iron storage protein ferritin is regulated reciprocally at the post-transcriptional level according to the cellular iron status. As a result of externalization of TfR during the endocytic cycle, a soluble form of TfR can be detected in serum. The serum TfR (sTfR) level is closely related to erythroid TfR turnover and the prime determinants of the sTfR concentration are cellular iron demands and erythroid proliferation rate. In the absence of a hyperplastic erythropoiesis the sTfR level is a sensitive parameter of early tissue iron deficiency. The entire spectrum of body iron status can be assessed by measurement of serum ferritin and sTfR levels, with ferritin as marker of tissue iron stores and sTfR as index of tissue iron needs. The sTfR may be a promising tool to detect iron deficiency in inflammatory states and in the anaemia of chronic disease as its concentration is, in contrast to ferritin levels, not influenced by the acute phase response. Determination of sTfR levels may also improve assessment of body iron stores during pregnancy and in neonates. Finally, the sTfR may be a useful parameter to monitor erythropoiesis in various clinical settings, for instance in the prediction of the haematological response to erythropoietin treatment. However, standardization of the sTfR assay, with definition of reference and pathological ranges, is necessary for the definitive introduction of the sTfR as major parameter of iron metabolism.

Non-transferrin iron uptake by trophoblast cells in culture. Significance of a NADH-dependent ferrireductase.

This study shows that trophoblast cells in culture are able to take up 59Fe from both Fe(III)nitrilotriacetate (NTA) and Fe-ascorbate. Fe in the presence of ascorbate is assumed to be Fe(III) in equilibrium with Fe(II). Kinetic parameters for non-transferrin iron uptake are determined from initial rate experiments, yielding Vmax=366 pmol/mg protein/5 min and Km=0.96 microM for Fe(III)NTA and Vmax=4043 pmol/mg protein/5 min and Km= 1.3 microM for Fe-ascorbate. Since trophoblast cells in culture reduce extracellular Fe(III)CN, and uptake of 59Fe from Fe-ascorbate is higher than that from Fe(III)NTA, it is suggested that reduction of Fe(III) precedes uptake. Uptake of 59Fe from both Fe-ascorbate and Fe(III)NTA is inhibited by Fe(II)chelator ferrozine and membrane-impermeable Fe(III)CN, further supporting this hypothesis. Studies with microvillous membrane vesicles (MMV) and basal membrane vesicles (BMV) reveal the presence of a NADH-dependent ferrireductase. Reduction of Fe(III)CN follows Michaelis-Menten kinetics, both with respect to [NADH] and [Fe]. NADPH is ineffective as electron donor. The rate of Fe(III)CN reduction by BMV is 2.5 times higher compared to MMV, while Km values for Fe(III)CN and NADH are not significantly different. These results reveal that a transmembrane NADH-dependent ferrireductase plays a role in uptake of non-transferrin iron. The possibility that this enzyme system is involved in iron transfer across the basal membrane is discussed.

Regulation of iron metabolism in the acute-phase response: interferon gamma and tumour necrosis factor alpha induce hypoferraemia, ferritin production and a decrease in circulating transferrin receptors in cancer patients.

BACKGROUND: The acute-phase response and anaemia of chronic disease are characterized by hypoferraemia associated with an increased ferritin synthesis, which might be mediated by the activated cytokine cascade. METHODS: We examined the prolonged effects of isolated limb perfusion (ILP) with recombinant human tumour necrosis factor alpha (rTNF), recombinant human interferon gamma (rIFN-gamma) and melphalan on interleukin (IL) 6 and acute-phase protein levels, iron status and serum transferrin receptor (sTfR) levels in 12 patients with melanoma or sarcoma. Patients were treated with ILP during 90 min after pretreatment with rIFN-gamma during 2 days. RESULTS: After ILP, leakage of TNF resulted in systemic peak levels at 3 min followed by an increase in IL-6 with maximum levels at 4h. C-reactive protein (CRP) rose at 4 h to peak levels at day 2, whereas alpha 1-antitrypsin and alpha 1-acid glycoprotein increased to maximum levels at day 3. Albumin and transferrin levels decreased after ILP and recovered after day 2. Serum iron and sTfR levels decreased during pretreatment and after ILP to minimum levels at 8 h and day 1 respectively. This was associated with an increase in serum ferritin levels, which paralleled CRP values. CONCLUSIONS: Our data point to a central role for the cytokine network in the modulation of iron metabolism in the acute-phase response and anaemia of chronic disease. TNF, possibly via induction of IL-6, and IFN-gamma induce hypoferraemia, which may in part result from a decrease in tissue iron release based on a primary stimulation of ferritin synthesis. The fall in sTfR levels may reflect an impaired erythroid growth and/or TfR expression mediated by TNF and IFN-gamma.

Iron uptake in blood-brain barrier endothelial cells cultured in iron-depleted and iron-enriched media.

Iron is essential in the cellular metabolism of all mammalian tissues, including the brain. Intracerebral iron concentrations vary with age and in several (neurological) diseases. Although it is evident that endothelial cells lining the capillaries in the brain are of importance, factors governing the regulation of intracerebral iron concentration are unknown. To investigate the role of blood-brain barrier endothelial cells in cerebral iron regulation, primary cultures of porcine blood-brain barrier endothelial cells were grown in either iron-enriched or iron-depleted medium. Iron-enriched cells showed a reduction in surface-bound and total transferrin receptor numbers compared with iron-depleted cells. Transferrin receptor kinetics showed that the transferrin receptor internalization rate in iron-enriched cultures was higher, whereas the transferrin receptor externalization rate in iron-enriched cultures was lower than the rate in iron-depleted cultures. Moreover, blood-brain barrier endothelial cells cultured in iron-enriched medium were able to accumulate more iron intracellularly, which underlines our kinetic data on transferrin receptors. Our results agree with histopathological studies on brain tissue of patients with hemochromatosis, suggesting that at high peripheral iron concentrations, the rate of iron transport across the blood-brain barrier endothelial cells is to some extent proportional to the peripheral iron concentration.

Evaluation of the iron status of the newborn by soluble transferrin receptors in serum.

The concentration of soluble transferrin receptors in serum has proven to be a reliable predictor of iron status in adults. Its high sensitivity for iron deficiency combined with a small sample size (10 microliters) makes it an interesting parameter for the assessment of iron stores in newborn infants. In the present study we investigated the usefulness of the concentration of soluble transferrin receptors in serum in the assessment of iron metabolism in the newborn. Infants born after an uncomplicated labour were compared to infants in the intensive care unit. The concentration of soluble transferrin receptors in serum was found to be elevated compared to normal adults and independently of iron metabolism. The concentration of soluble transferrin receptors did not correlate with serum iron and ferritin concentrations. In contrast to what was found in other studies, no relationship could be demonstrated between soluble transferrin receptors and birth weight or gestational age. The results of this study have shown that care has to be taken in the interpretation of the concentration of soluble transferrin receptors in serum in newborn infants. It seems to be a parameter which is independent of iron metabolism at least during the first days of life.

Relationship between soluble transferrin receptors in serum and membrane-bound transferrin receptors.

The expression of transferrin receptors on the cell membrane of erythroblasts was analysed with flow cytometry in patients with different forms of anaemia. At the same time the concentration of soluble transferrin receptors (sTfRs) was analysed in serum. It was shown that only in iron deficiency a high concentration of sTfRs in serum could be explained with an increased expression of transferrin receptors on the erythroblastic membrane. In anaemia of chronic disease and myelodysplasia a discrepancy between a low expression on the cell membrane and normal or elevated serum values was seen. From this study we conclude that the concentration of sTfRs in serum does not only depend on the expression of transferrin receptors on the erythroblasts but also on the erythroid activity.

Transferrin in cultured human term cytotrophoblast cells: synthesis and heterogeneity.

Transferrin (Tf) mRNA was recently demonstrated in rat and mouse placental tissue. Rat placental cells were shown to secrete transferrin. The cell type with which Tf mRNA was associated was not investigated. We therefore studied the ability of immunopurified human term cytotrophoblast cells in culture to synthesize Tf, by means of pulse-label experiments with 35S-methionine and report that these cells do synthesize Tf. Tf mRNA was demonstrated in the cell lysates by means of RT-PCR. Tf isolated from cytotrophoblast and syncytiotrophoblast cells was shown to be different from both maternal and fetal serum Tf with respect to the distribution of isoforms as demonstrated by means of iso-electric focusing. The iso-electric points were found at lower pH values (pH 5.0-5.4), compared to the iso-electric points of maternal and fetal serum Tf, suggesting a higher degree of sialylation and glycan chain complexity.

Isoforms of transferrin in psoriasis patients abusing alcohol.

The different isoforms of transferrin have been quantified by isoelectric focusing in the sera of psoriasis patients with and without a history of abusing alcohol. In both male and female psoriasis subjects abusing alcohol, there were significant increases in the 2-sialylated forms by comparison to the control subjects. Psoriasis patients who had no evidence of alcohol abuse had similar profile for the isoforms of transferrin to that of the controls. Other groups of patients with alcohol-induced tissue damage, i.e. liver, brain or muscle, used as positive controls, similarly showed significant increases in the 2-sialylated forms, by comparison to controls. These results substantiate the current use of carbohydrate-deficient transferrin as a sensitive marker of alcohol abuse, particularly in subjects not drinking in excess of 60 g of ethanol/day but showing alcohol-related psoriasis.

Influence of transferrin glycans on receptor binding and iron-donation.

Human bi-bi-antennary transferrin (Tf) was partially deglycosylated by subsequently incubating with one or more of the following exoglycosidases: neuraminidase, beta-galactosidase or N-Acetyl-beta-D-glucosaminidase. Aglyco-Tf obtained from serum of a patient suffering from the Carbohydrate Deficient Glycoprotein syndrome was isolated. Receptor binding and the Tf and iron uptake capacities of the fully glycosylated-, partially deglycosylated- and aglyco-Tf were compared using the human hepatoma cell line PLC/PRF/5. No difference in binding capacity between the iso-Tf fractions could be demonstrated, however, the Tf and iron uptake capacity of aglyco-Tf was clearly reduced compared with the other Tf fractions.

Transcytosis of 6.6-nm gold-labeled transferrin: an ultrastructural study in cultured porcine blood-brain barrier endothelial cells.

The mechanism and regulation of iron transport to the brain are largely unknown. The large surface area of the blood-brain barrier capillaries and the presence of transferrin receptors on the luminal plasma membranes of the blood-brain barrier endothelial cells (BBB-ECs) suggest that these cells actively participate in the transport of iron into the brain. In this paper, we describe the ultrastructural morphology of primary and first-passage cultures of BBB-ECs grown on different types of porous membranes. To investigate the mechanism of iron transport into and across the BBB-ECs, porous membrane grown first-passage cells were incubated with 6.6-nm gold-labeled transferrin and studied with electron microscopy. Results are suggestive for a transcytosis of transferrin through the BBB-ECs.

Binding of human isotransferrin variants to microvillous and basal membrane vesicles from human term placenta.

Transferrin (Tf)-dependent iron transfer from mother to fetus is mediated by Tf receptors (TfRs) which are present on both microvillous and basal membranes of human placental syncytiotrophoblast. We used microvillous and basal membrane vesicles, both isolated from the same human term placenta, to investigate the binding of [125I]-labelled diferric bi-bi antennary tetra-sialo Tf (bb Tf), bi-tri-antennary penta-sialo Tf (bt Tf) and tri-tri-antennary hexa-sialo Tf (tt Tf). To diminish the effect of endogenous Tf, membrane vesicles were washed before binding of [125I]-Tf. The number of TfRs on microvillous membranes was 6.1 +/- 2.4 (mean +/- s.d., n = 15) times higher than that on basal membranes, whereas the affinity of TfRs on basal membranes was 3.9 +/- 0.4 (mean +/- s.d., n = 15) times higher than that of TfRs on microvillous membranes, irrespective the isoTf used. The affinity constants of TfRs on both microvillous and basal membranes were higher for bb Tf than for bt Tf and higher for bt Tf than for tt Tf. However, these latter differences were rather small and probably not of physiological importance.

The expression of transferrin receptors on erythroblasts in anaemia of chronic disease, myelodysplastic syndromes and iron deficiency.

The presence of transferrin receptors on erythroblasts in patients with iron deficiency, anaemia of chronic disease (ACD) and myelodysplastic syndrome (MDS) was studied by two-colour analysis on a flow cytometer. CD 71 was used to quantify the number of transferrin receptors and GLY-A to identify erythroblasts. In cases of iron deficiency, the number of transferrin receptors was increased on part of the erythroblasts thus facilitating iron uptake by the cells. In patients with ACD or MDS, a decrease of the number of transferrin receptors on erythroblasts was found. This leads to the conclusion that the ineffective response to iron therapy in cases of ACD and MSD can be explained by a decline of transferrin receptors on the red cells.

New developments and applications in quantitative electron spectroscopic imaging of iron in human liver biopsies.

Reliable iron concentration data can be obtained by quantitative analyses of image sequences, acquired by electron spectroscopic imaging. A number of requirements are formulated for the successful application of this recently developed in situ quantitative type of analysis. A demonstration of the procedures is given. By application of the technique it is established that there are no significant differences in the average iron loading of structures analysed in liver parenchymal cells of a patient with an iron storage disease, before and after phlebotomy. This supports the hypothesis that the process of iron unloading is an organelle specific process. Measurement of the binary morphology, represented by the area and contour ratio of the iron containing objects revealed no information about differences between the objects. This finding contradicts the visual suggestion that ferritin clusters are more irregularly shaped than the other iron objects. Also, no differences could be found in this sense between the situations before and after phlebotomy. With respect to the density appearance, objects that have an inhomogeneous iron loading averagely contain more iron. This observation does correspond well with the visual impression of the increasingly irregular appearance of more well-loaded structures.

Isolation and partial characterization of two porcine spleen ferritin fractions with different electrophoretic mobility.

Ferritin isolated from porcine spleen could routinely be separated in two fractions on nondenaturating gradient gels. Both fractions could be isolated with a purity of 96% when applied to two serially linked columns, each 200 cm in length, packed respectively with Sepharose 4B and Sepharose 6B. Both fractions were similar as judged by electron microscopy. Assessed biochemically fractions were equal with respect to subunit composition, iron and phosphorus content, as well as amino acid composition (with the exception of N-acetylglucosamine). Carbohydrate analysis showed that the fraction with an apparent mass of 440 kDa (= FFL) contained 1.8% (w/w) glycans, whereas the fraction with an apparent mass of 670 kDa (= FFH) contained nearly five times as much (neutral) sugar residues (8.9%, w/w) and 10 times as much sialic acid. This difference in amount of carbohydrate side chains might explain the dissimilarity in electrophoretic mobility of the two fractions.

Isolation, renaturation and partial characterization of recombinant human transferrin and its half molecules from Escherichia coli.

Recombinant human transferrin as well as N- and C-terminal half-transferrins, produced in Escherichia coli, are deposited in inclusion bodies by the bacteria. The isolation and purification of the recombinant proteins from these inclusion bodies are described here. The amino acid compositions and N-terminal sequences of the proteins were determined, and found to be in agreement with the known protein structure of human serum transferrin. Renaturation of the recombinant proteins is described, resulting in water-soluble iron-binding molecules. Iron binding was confirmed by 59Fe labelling, absorption spectrophotometry and EPR spectrometry.

Analytical and clinical implications of soluble transferrin receptors in serum.

In this study the analytical performances of two recently introduced assays for soluble transferrin receptors in serum were tested. The Ramco transferrin assay was compared with the Eurogenetics assay. In a small clinical study serum samples from patients with anaemia of chronic disease, iron deficiency and myelodysplastic syndrome were analysed, as well as sera from healthy volunteers. The analytical performances of the Ramco assay were found to be acceptable. In the Eurogenetics test however, inter-assay imprecision and the end of run drift were unacceptably high. We were able to confirm that in patients with uncomplicated iron deficiency the concentration of soluble transferrin receptors is higher than in healthy volunteers. In cases of anaemia of chronic and inflammatory disease, the levels of soluble transferrin receptors in serum are slightly, but not significantly, higher than in normal subjects. Measurement of soluble transferrin receptors in serum provides a good differentiation between anaemia of chronic disease and iron deficiency.