Iron in the General Population and Specificities in Older Adults: Metabolism, Causes and Consequences of Decrease or Overload, and Biological Assessment.

Iron is involved in many types of metabolism, including oxygen transport in hemoglobin. Iron deficiency (ID), ie a decrease in circulating iron, can have severe consequences. We provide an update on iron metabolism and ID, highlighting the particularities in older adults (OAs). There are three iron compartments in the human body: 1) the functional compartment, which consists of heme proteins including hemoglobin, myoglobin and respiratory enzymes; 2) iron reserves (IR), which consist mainly of liver stocks and are stored as ferritin; and 3) transferrin. There are two types of ID. Absolute ID is characterized by a decrease in IR. Its main pathophysiological mechanism is bleeding, which is often digestive and can be due to neoplasia, frequent in OAs. Biological assessment shows low serum ferritin and transferrin saturation (TS) levels. Furthermore, hypochromic microcytic anemia is frequent, and the serum-soluble transferrin receptor (sTfR) level is high. Functional ID, in which IR are high or normal, is due to inflammation, which is also frequent in OAs, particularly in its chronic form. Biological assessments show high serum ferritin, normal or low TS, and normal sTfR levels. Moreover, C-reactive protein is elevated, and there is moderate non-regenerative non-macrocytic anemia. The main characteristics of iron metabolism anomalies in the elderly are the high frequency of ID (20% of ID with anemia in adults ≥85 years) and the severity of its consequences, which include cognitive impairment in case of ID or iron overload and decrease of physical activity in case of ID. In conclusion, causes of ID are frequently intertwined in OAs as a result of the polymorbidity that characterizes them. ID can have dramatic consequences, especially in frail OAs. Thus, measuring the appropriate biological markers prevents errors in the positive diagnosis of ID type, clarifies etiology, and informs treatment-related decision-making.

A liquid chromatography-tandem mass spectrometry-based targeted proteomics approach for the assessment of transferrin receptor levels in breast cancer.

PURPOSE: Overexpression of human transferrin receptor (TfR) has been described qualitatively in various cancers, including breast cancer. Since TfR is also expressed to some extent under normal physiological conditions, increase of specificity and reproducibility in TfR quantification could improve the early detection and prognostic evaluation of cancers. EXPERIMENTAL DESIGN: A LC-MS/MS-based targeted proteomics assay was developed for the determination of TfR in human breast cells and tissue samples. RESULTS: We selected the tryptic peptide 681VEYHFLSPYVSPK693 as the surrogate peptide for quantification and used a stable isotope-labeled synthetic peptide with this sequence as an internal standard. Quality control data indicated acceptable accuracy and precision. Finally, this assay was successfully applied to the quantitative analysis of TfR in three breast cell lines and 36 matched pairs of breast tissue samples. The experimental values were compared with those obtained from conventional analytical methods, including immunofluorescence microscopy, Western blotting, flow cytometry, and immunohistochemistry. CONCLUSIONS AND CLINICAL RELEVANCE: Not only is the LC-MS/MS-based targeted proteomics assay a more accurate method for the determination of TfR levels, but may afford more reliable quantification of TfR at low concentrations in clinical practice.

Assessment of the roles of ordered lipid microdomains in post-endocytic trafficking of glycosyl-phosphatidylinositol-anchored proteins in mammalian fibroblasts.

We have used artificial phosphatidylethanolamine-polyethylene glycol (PE-PEG)-anchored proteins, incorporated into living mammalian cells, to evaluate previously proposed roles for ordered lipid 'raft' domains in the post-endocytic trafficking of glycosylphosphatidylinositol (GPI)-anchored proteins in CHO and BHK cells. In CHO cells, endocytosed PE-PEG protein conjugates colocalized strongly with the internalized GPI-anchored folate receptor, concentrating in the endosomal recycling compartment, regardless of the structure of the hydrocarbon chains of the PE-PEG 'anchor'. However, internalized PE-PEG protein conjugates with long-chain saturated anchors recycled to the plasma membrane at a slow rate comparable to that measured for the GPI-anchored folate receptor, whereas conjugates with short-chain or unsaturated anchors recycled at a faster rate similar to that observed for the transferrin receptor. These findings support the proposal (Mayor et al. Cholesterol-dependent retention of GPI-anchored proteins in endosomes. EMBO J 1998;17:4628-4638) that the slow recycling of GPI proteins in CHO cells rests on their affinity for ordered lipid domains. In BHK cells, internalized PE-PEG protein conjugates with either saturated or unsaturated 'anchors' colocalized strongly with simultaneously endocytosed folate receptor and, like the folate receptor, gradually accumulated in late endosomes/lysosomes. These latter findings do not support previous suggestions that the sorting of GPI proteins to late endosomes in BHK cells depends on their association with lipid rafts.

The assessment of human erythroid output in NOD/SCID mice reconstituted with human hematopoietic stem cells.

The third-generation NOD/LtSz-scid/IL2Rγ(null) (NOD/SCID IL2Rγ(null)) mouse represents a significantly improved xenograft model allowing high levels of human leukocyte engraftment over extended follow up. One remaining limitation of this mouse model, however, is the low level of circulating human erythrocytes. We established a practical ex vivo erythroid culture system of xenograft marrow progenitors to enrich for human erythroid progeny. At various time points after transplant, erythroid cells were easily assayed after 17 days of ex vivo culture of xenograft marrow, with nearly all nucleated cells of human origin and approximately 60% human GPA or CD71 positive. We then transplanted cord blood CD34(+) cells marked with a lentiviral vector encoding green fluorescent protein (GFP). Three months later, ex vivo culture of xenograft marrow progenitors showed 41.3% of the cultured erythroid cells were positive for GFP and human CD71, and 56.2% were positive for GFP and human GPA, similar to that of circulating leukocytes at the same time point. Next, G-CSF mobilized peripheral blood CD34(+) cells from a sickle cell trait subject were infused in this mouse model to determine if the hemoglobin pattern could be modeled. CD34(+) cells from the sickle cell trait subject engrafted equally compared to CD34(+) cells from normal subjects, establishing the sickle cell trait phenotype. Lastly, a comparison of adult-derived peripheral blood CD34(+) cells and cord blood-derived CD34(+) cells xenografted mice was made, and long term follow-up demonstrated a recapitulation of the fetal to adult hemoglobin switch. This approach should prove a useful tool for testing strategies for genetic manipulation of erythroid progeny and the study of hemoglobin switching.

[The comparison of diagnostic usefulness of transferrin determination by immunological and chemical method in iron deficiency anemia]. / Porównanie przydatnosci diagnostycznej immunologicznej i chemicznej metody oznaczania stezenia transferyny w niedokristosci z powodu niedoboru zelaza.

UNLABELLED: The aim of this study was to compare the diagnostic accuracy of two methods of transferrin determination in iron-deficiency anemia (IDA): immunological and chemical based on the total iron-binding capacity (TIBC). MATERIAL AND METHODS: The examination was carried out in 60 women with IDA and 20 healthy controls. Anemia was defined as a hemoglobin concentration less than 120 g/l, ferritin level below 30 microg/l and clinical date. RESULTS: In female IDA the transferrin level measured by immunological method was significantly higher than that evaluated by chemical procedure. In control subjects the results were not significantly different. The serum transferrin concentration measured by these two methods correlated positively each other. The diagnostic sensitivity, specificity, efficiency, positive and negative predictive values were higher for immunological methods. The diagnostic accuracy of immunological methods (area under the curve - AUC) did not differ in comparison to the chemical methods when calculated from empirical coefficient (value 22.3). CONCLUSION: Calculation at the theoretical coefficient (value 25.0), the diagnostic accuracy of immunological method was higher than that of chemical. The good points of immunological methods of transferrin determination (direct, simplicity, higher precision, un-dependent on iron binding, higher sensitivity) should be taken into account for the introduction of this method for routine laboratory diagnostics instead of chemical methods based on total iron-binding capacity.

In vitro assessment of transferrin-conjugated liposomes as drug delivery systems for inhalation therapy of lung cancer.

Most human tumours over-express receptors for growth factors and peptide hormones, which are being increasingly studied as a means to selectively deliver cytotoxic agents. An example being the transferrin receptor (TfR, CD71). Here, we studied expression levels and location of TfR in different lung epithelial cell types (i.e., bronchial and alveolar epithelial cells) by flow-cytometry and confocal laser scanning microscopy (CLSM). Furthermore, we assessed uptake levels and cytotoxicity of transferrin (Tf)-conjugated liposomes in vitro. TfR was found to be expressed at a significantly higher level in bronchial epithelial cells compared with their alveolar counterparts. Cells of cancerous origin (i.e., A549 cell line) showed a higher TfR expression level than healthy alveolar epithelial type II cells in primary culture. CLSM revealed TfR to be located primarily at the basolateral aspect of cells, with the exception of cells undergoing mitotic proliferation, which also showed TfR at their apical membranes, due to their loss of cell polarity. Higher expression levels of TfR correlated well with enhanced uptake of Tf-liposomes and increased levels of cytotoxicity. Liposome uptake was temperature-dependent and inhibitable by excess free Tf. Tf-conjugated liposomes appear as good candidates for an approach to deliver cytostatic drugs to sites of lung cancer by inhalation.

Endocytic internalization of adenovirus, nonspecific phagocytosis, and cytoskeletal organization are coordinately regulated in alveolar macrophages by GM-CSF and PU.1.

GM-CSF gene-targeted (GM(-/-)) mice have impaired pulmonary clearance of bacterial and fungal pathogens by alveolar macrophages (AMs). Because AMs also clear adenovirus from the lung, the role of GM-CSF in endocytic internalization of adenovirus by AMs was evaluated. Pulmonary clearance of adenovirus was severely impaired in GM(-/-) mice compared to wild-type (GM(+/+)) mice as determined by Southern analysis of viral DNA. Internalization of adenovirus by AMs was deficient in GM(-/-) mice in vivo and in vitro as determined by uptake of fluorescently labeled adenovirus or by PCR quantification of adenoviral DNA internalized within AMs. An AM cell line previously established from GM(-/-) mice (mAM) had impaired internalization of adenovirus and transferrin-coated 100-nm latex beads compared to MH-S, a GM(+/+) AM cell line. Phagocytosis of 4- micro m latex beads was also impaired in mAM cells as determined by confocal and fluorescence microscopy. Retroviral vector-mediated reconstitution of PU.1 expression in cultured GM(-/-) AMs restored phagocytosis of 4- micro m beads, endocytosis of adenovirus, and transferrin-coated 100-nm beads (independent of integrin alpha(V) and transferrin receptors, respectively), and restored normal cytoskeletal organization, filamentous actin distribution, and stimulated formation of filopodia. Interestingly, mRNA for the phosphoinositide 3 kinase p110gamma isoform, important in macrophage phagocytic function, was absent in GM(-/-) AMs and was restored by PU.1 expression. These data show that GM-CSF, via PU.1, regulates endocytosis of small ( approximately 100 nm) pathogens/inert particles and phagocytosis of very large inert particles and suggests regulation of cytoskeletal organization by GM-CSF/PU.1 as the molecular basis of this control.

Assessment of erythropoiesis activity during hemodialysis therapy by soluble transferrin receptor levels and ferrokinetic measurements.

The erythropoietic activity (EA) and degree of erythropoiesis attained by patients undergoing hemodialysis (HD) administered recombinant human erythropoietin (rHuEPO) were studied using ferrokinetic measurements and tests of soluble transferrin receptor (sTfR) levels, assessing which parameter is most useful for measurements in clinical practice. Plasma iron 59 ((59)Fe) clearance (half-life [T(1/2)] (59)Fe), plasma iron turnover (PIT), erythron transferrin uptake (ETU), and erythrocyte (59)Fe incorporation were determined in 23 patients before and at 4 months after administration of rHuEPO. sTfR levels, hematopoietic parameters, and iron metabolism parameters were measured periodically. T(1/2) (59)Fe was shortened (P: = 0.004), PIT and ETU were increased (P: = 0.032 and P: = 0.013, respectively), and the time taken by erythrocytes to incorporate 80% of the (59)Fe administered was reduced from 9.6 to 6.1 days. sTfR levels were increased by 15 days; this increase was significant (P: < 0.05) at 30 days, reaching a maximum of 3.22 mg/dL at day 45. A positive correlation was seen between sTfR levels and hemoglobin (Hb) (P: = 0.001), hematocrit (P: = 0.001), and reticulocytes (P: = 0.038) that was not found between ferrokinetic parameters and those evaluating efficient erythropoiesis (P: = 0.345 between ETU and Hb). In conclusion, EA is increased, shown by ETU and sTfR level. sTfR levels correlate with the parameters that evaluate efficient erythropoiesis, and their measurement does not involve the technical and/or ethical limitations of studies of ferrokinetics, making them the tool of choice in clinical practice for the evaluation of EA in patients undergoing HD administered rHuEPO.

Quantitative assessment of erythropoiesis in haemodialysis patients demonstrates gradual expansion of erythroblasts during constant treatment with recombinant human erythropoietin.

Recombinant human erythropoietin (rHuEpo) has been shown to be effective in correcting the anaemia of chronic renal failure. It has been reported that reticulocytes as well as erythroid progenitors increase within 1-2 weeks, with no further elevation beyond this time interval. However, the erythroblast pool is quantitatively the most important compartment of erythropoiesis, and the rate, extent and duration of the expansion of erythropoietic activity in response to rHuEpo is not known. Treatment with rHuEpo was given to 64 patients i.v. thrice weekly after haemodialysis. The effect of rHuEpo was obvious from the early elevation of reticulocyte counts, but much of this increase was due to a rapid output of shift reticulocytes which levelled off after a few weeks. Serum transferrin receptor (TfR), a quantitative measure of erythropoiesis, increased progressively over 6 weeks to reach a plateau phase at about twice baseline values. The Hct increased progressively and continued to rise steadily after the TfR plateau was reached. The speed and extent of the expansion of erythropoietic activity correlated with the later haematological response to rHuEpo. When rHuEpo was discontinued, erythropoietic activity returned progressively to baseline values, to rise again gradually when treatment was resumed. Part of the Hct increase was also due to haemoconcentration. The results indicate that changes in the various erythroid compartments vary considerably in intensity and speed, and that the erythroblast compartment in particular is slow to respond to modifications in the erythropoietin stimulus.

Assessment of iron status of Zairean women of childbearing age by serum transferrin receptor.

Soluble transferrin receptor (sTfR), previously shown to be a sensitive indicator of tissue iron deficiency, was used to assess iron status of 104 Zairean women (69 lactating, 19 pregnant, and 16 nonpregnant, nonlactating women). Thirteen iron-sufficient female laboratory employees were also studied. Mean sTfR concentrations were higher in pregnant women (9.90 mg/L) than in lactating women (8.55 mg/L), nonlactating women (7.74 mg/L), and laboratory employees (5.11 mg/L) (P < 0.005). Mean serum ferritin and transferrin saturation were lower in pregnant than nonpregnant women. sTfR negatively correlated with hemoglobin (P < 0.05) and transferrin saturation (P < 0.05), and nonsignificantly with ferritin and transferrin. With 7.26 mg sTfR/L (the upper limit of laboratory employees) as the cutoff value, sTfR identified 67% of women with tissue iron deficiency compared with 11.5-57% for transferrin saturation, hemoglobin, or ferritin. Despite the moderate sensitivity (68.5%), 90% of women with sTfR > 7.26 mg/L also had another index below normal and 54% had serum ferritin < or = 50 micrograms/L.

Disparity between expression of transferrin receptor ligand binding and non-ligand binding domains on human lymphocytes.

We compared transferrin receptor (TfR) expression on human peripheral blood lymphocytes (PBL) activated by phorbol myristate acetate (PMA) or L-phytohemagglutinin (LPHA) using two techniques: (1) 125I-iron-saturated transferrin (FeTf) binding, (2) reactivity with monoclonal anti-TfR antibodies--OKT9 and B3/25. These monoclonal antibodies do not block FeTf binding, and therefore bind to TfR domains separate from the ligand binding site. Unstimulated PBL bound fewer than 1,000 molecules of 125I-FeTf per cell, and less than 5% of cells expressed TfR antigens detected by OKT9 or B3/25. 125I-FeTf binding and antibody binding increased in parallel on LPHA-activated PBL. After exposure to LPHA for 72 hr, 125I-FeTf binding increased 100-fold to 10(5) molecules per cell and greater than 50% of cells expressed TfR antigens. By contrast, PMA activation of PBL markedly increased binding of OKT9 and B3/25 but not the binding of 125I-FeTf. Cell surface expression of TfR antigens seen by OKT9 and B3/25 did not differ between LPHA- and PMA-activated PBL. However, after 72 hr with PMA, 125I-FeTf binding increased only 6-fold and consistently remained at less than 10(4) molecules per cell. Therefore, PMA induced a disparity between expression of TfR ligand binding domains and immunological domains at the cell surface. Cell proliferation assessed by fluorescent DNA analysis was similar in cultures stimulated by LPHA or PMA. These data indicate that lymphoid cells may possess a mechanism for modulating TfR expression in which down-regulation of FeTf binding occurs without receptor internalization. Alternatively, it is possible that this observation may reflect a membrane perturbation effect of PMA.