Fluorescence assay of non-transferrin-bound iron in thalassemic sera using bacterial siderophore

Transfusional iron overload associated with thalassemia leads to the appearance of non-transferrin-bound iron (NTBI) in blood that is toxic and causes morbidity and mortality via tissue damage. Hence, a highly sensitive and accurate assay of NTBI, with broad clinical application in both diagnosis and validation of treatment regimens for iron overload, is important. An assay based on iron chelation by a high-affinity siderophore, azotobactin, has been developed. The steps consist of blocking of native apotransferrin iron binding sites, mobilization of NTBI, ultrafiltration of all serum proteins, and finally the addition of the probe, which has a chromophore that fluoresces at 490 nm. Binding of Fe³⁺ to azotobactin quenches the fluorescence in a concentration-dependent manner. Measured NTBI levels in 63 sera ranged from 0.07 to 3.24 μM (0.375 ± 0.028 μM [means ± SEM]). It correlated well with serum iron and percentage transferrin saturation but not with serum ferritin. Pearson’s correlation coefficients were found to be 0.6074 (P < 0.0001) and 0.6102 (P < 0.0001) for percentage transferrin saturation and total serum iron, respectively. The low values are due to the patients being under regular chelation therapy even prior to sampling, indicating that the method is sensitive to very low levels of NTBI, allowing a much lower detection limit than the available methods.     

Iron loading induces cholesterol synthesis and sensitizes endothelial cells to TNFα-mediated apoptosis

In plasma, iron is normally bound to transferrin, the principal protein in blood responsible for binding and transporting iron throughout the body. However, in conditions of iron overload when the iron-binding capacity of transferrin is exceeded, non–transferrin-bound iron (NTBI) appears in plasma. NTBI is taken up by hepatocytes and other parenchymal cells via NTBI transporters and can cause cellular damage by promoting the generation of reactive oxygen species. However, how NTBI affects endothelial cells, the most proximal cell type exposed to circulating NTBI, has not been explored. We modeled in vitro the effects of systemic iron overload on endothelial cells by treating primary human umbilical vein endothelial cells (HUVECs) with NTBI (ferric ammonium citrate [FAC]). We showed by RNA-Seq that iron loading alters lipid homeostasis in HUVECs by inducing sterol regulatory element-binding protein 2–mediated cholesterol biosynthesis. We also determined that FAC increased the susceptibility of HUVECs to apoptosis induced by tumor necrosis factor-α (TNFα). Moreover, we showed that cholesterol biosynthesis contributes to iron-potentiated apoptosis. Treating HUVECs with a cholesterol chelator hydroxypropyl-β-cyclodextrin demonstrated that depletion of cholesterol was sufficient to rescue HUVECs from TNFα-induced apoptosis, even in the presence of FAC. Finally, we showed that FAC or cholesterol treatment modulated the TNFα pathway by inducing novel proteolytic processing of TNFR1 to a short isoform that localizes to lipid rafts. Our study raises the possibility that iron-mediated toxicity in human iron overload disorders is at least in part dependent on alterations in cholesterol metabolism in endothelial cells, increasing their susceptibility to apoptosis.     

Prion Protein Promotes Kidney Iron Uptake via Its Ferrireductase Activity

Brain iron-dyshomeostasis is an important cause of neurotoxicity in prion disorders, a group of neurodegenerative conditions associated with the conversion of prion protein (PrP^C) from its normal conformation to an aggregated, PrP-scrapie (PrP^Sc) isoform. Alteration of iron homeostasis is believed to result from impaired function of PrP^C in neuronal iron uptake via its ferrireductase activity. However, unequivocal evidence supporting the ferrireductase activity of PrP^C is lacking. Kidney provides a relevant model for this evaluation because PrP^C is expressed in the kidney, and ∼370 μg of iron are reabsorbed daily from the glomerular filtrate by kidney proximal tubule cells (PT), requiring ferrireductase activity. Here, we report that PrP^C promotes the uptake of transferrin (Tf) and non-Tf-bound iron (NTBI) by the kidney in vivo and mainly NTBI by PT cells in vitro. Thus, uptake of ⁵⁹Fe administered by gastric gavage, intravenously, or intraperitoneally was significantly lower in PrP-knock-out (PrP^−/−) mouse kidney relative to PrP^+/+ controls. Selective in vivo radiolabeling of plasma NTBI with ⁵⁹Fe revealed similar results. Expression of exogenous PrP^C in immortalized PT cells showed localization on the plasma membrane and intracellular vesicles and increased transepithelial transport of ⁵⁹Fe-NTBI and to a smaller extent ⁵⁹Fe-Tf from the apical to the basolateral domain. Notably, the ferrireductase-deficient mutant of PrP (PrP^Δ51–89) lacked this activity. Furthermore, excess NTBI and hemin caused aggregation of PrP^C to a detergent-insoluble form, limiting iron uptake. Together, these observations suggest that PrP^C promotes retrieval of iron from the glomerular filtrate via its ferrireductase activity and modulates kidney iron metabolism.     

Non-transferrin-bound iron uptake in Belgrade and normal rat erythroid cells

Belgrade (b) rats have an autosomal recessive, microcytic, hypochromic anemia. Transferrin (Tf)-dependent iron uptake is defective because of a mutation in DMT1 (Nramp2), blocking endosomal iron efflux. This experiment of nature permits the present study to address whether the mutation also affects non-Tf-bound iron (NTBI) uptake and to use NTBI uptake compared to Tf-Fe utilization to increase understanding of the phenotype of the b mutation. The distribution of ⁵⁹Fe²⁺ into intact erythroid cells and cytosolic, stromal, heme, and nonheme fractions was different after NTBI uptake vs. Tf-Fe uptake, with the former exhibiting less iron into heme but more into stromal and nonheme fractions. Both reticulocytes and erythrocytes exhibit NTBI uptake. Only reticulocytes had heme incorporation after NTBI uptake. Properly normalized, incorporation into b/b heme was ∼20% of +/b, a decrease similar to that for Tf-Fe utilization. NTBI uptake into heme was inhibited by bafilomycin A1, concanamycin, NH₄Cl, or chloroquine, consistent with the endosomal location of the transporter; cellular uptake was uninhibited. NTBI uptake was unaffected after removal of Tf receptors by Pronase or depletion of endogenous Tf. Concentration dependence revealed that NTBI uptake into cells, cytosol, stroma, and the nonheme fraction had an apparent low affinity for iron; heme incorporation behaved like a high-affinity process, as did an expression assay for DMT1. DMT1 serves in both apparent high-affinity NTBI membrane transport and the exit of iron from the endosome during Tf delivery of iron in rat reticulocytes; the low-affinity membrane transporter, however, exhibits little dependence on DMT1. J. Cell. Physiol. 178:349–358, 1999. © 1999 Wiley-Liss, Inc.     

Nature of non-transferrin-bound iron: studies on iron citrate complexes and thalassemic sera

Despite its importance in iron-overload diseases, little is known about the composition of plasma non-transferrin-bound iron (NTBI). Using 30-kDa ultrafiltration, plasma from thalassemic patients consisted of both filterable and non-filterable NTBI, the filterable fraction representing less than 10% NTBI. Low filterability could result from protein binding or NTBI species exceeding 30 kDa. The properties of iron citrate and its interaction with albumin were therefore investigated, as these represent likely NTBI species. Iron permeated 5- or 12-kDa ultrafiltration units completely when complexes were freshly prepared and citrate exceeded iron by tenfold, whereas with 30-kDa ultrafiltration units, permeation approached 100% at all molar ratios. A g = 4.3 electron paramagnetic resonance signal, characteristic of mononuclear iron, was detectable only with iron-to-citrate ratios above 1:100. The ability of both desferrioxamine and 1,2-dimethyl-3-hydroxypyridin-4-one to chelate iron in iron citrate complexes also increased with increasing ratios of citrate to iron. Incremental molar excesses of citrate thus favour the progressive appearance of chelatable lower molecular weight iron oligomers, dimers and ultimately monomers. Filtration of iron citrate in the presence of albumin showed substantial binding to albumin across a wide range of iron-to-citrate ratios and also increased accessibility of iron to chelators, reflecting a shift towards smaller oligomeric species. However, in vitro experiments using immunodepletion or absorption of albumin to Cibacron blue–Sepharose indicate that iron is only loosely bound in iron citrate–albumin complexes and that NTBI is unlikely to be albumin-bound to any significant extent in thalassemic sera.     

Non-transferrin-bound iron in plasma following administration of oral iron drugs

Non-transferrin-bound iron (NTBI) was detected in serum samples from volunteers with normal iron stores or from patients with iron deficiency anaemia after oral application of pharmaceutical iron preparations. Following a 100 mg ferrous iron dosage, NTBI values up to 9 μM were found within the time period of 1–4 h after administration whereas transferrin saturation was clearly below 100%. Smaller iron dosages (10 and 30 mg) gave lower but still measurable NTBI values. The physiological relevance of this finding for patients under iron medication has to be elucidated.     

Multidentate pyridinones inhibit the metabolism of nontransferrin-bound iron by hepatocytes and hepatoma cells.

The therapeutic effect of iron (Fe) chelators on the potentially toxic plasma pool of nontransferrin-bound iron (NTBI), often present in Fe overload diseases and in some cancer patients during chemotherapy, is of considerable interest. In the present investigation, several multidentate pyridinones were synthesized and compared with their bidentate analogue, deferiprone (DFP; L1, orally active) and desferrioxamine (DFO; hexadentate; orally inactive) for their effect on the metabolism of NTBI in the rat hepatocyte and a hepatoma cell line (McArdle 7777, Q7). Hepatoma cells took up much less NTBI than the hepatocytes (< 10%). All the chelators inhibited NTBI uptake (80-98%) much more than they increased mobilization of Fe from cells prelabelled with NTBI (5-20%). The hexadentate pyridinone, N,N,N-tris(3-hydroxy-1-methyl-2(1H)-pyridinone-4-carboxaminoethyl)amine showed comparable activity to DFO and DFP. There was no apparent correlation between Fe status, Fe uptake and chelator activity in hepatocytes, suggesting that NTBI transport is not regulated by cellular Fe levels. The intracellular distribution of iron taken up as NTBI changed in the presence of chelators suggesting that the chelators may act intracellularly as well as at the cell membrane. In conclusion (a) rat hepatocytes have a much greater capacity to take up NTBI than the rat hepatoma cell line (Q7), (b) all chelators bind NTBI much more effectively during the uptake phase than in the mobilization of Fe which has been stored from NTBI and (c) while DFP is the most active chelator, other multidentate pyridinones have potential in the treatment of Fe overload, particularly at lower, more readily clinically available concentrations, and during cancer chemotherapy, by removing plasma NTBI.     

Molecular mechanisms of non‐transferrin‐bound and transferring‐bound iron uptake in primary hippocampal neurons

The molecular mechanisms of iron trafficking in neurons have not been elucidated. In this study, we characterized the expression and localization of ferrous iron transporters Zip8, Zip14 and divalent metal transporter 1 (DMT1), and ferrireductases Steap2 and stromal cell‐derived receptor 2 in primary rat hippocampal neurons. Steap2 and Zip8 partially co‐localize, indicating these two proteins may function in Fe³⁺ reduction prior to Fe²⁺ permeation. Zip8, DMT1, and Steap2 co‐localize with the transferrin receptor/transferrin complex, suggesting they may be involved in transferrin receptor/transferrin‐mediated iron assimilation. In brain interstitial fluid, transferring‐bound iron (TBI) and non‐transferrin‐bound iron (NTBI) exist as potential iron sources. Primary hippocampal neurons exhibit significant iron uptake from TBI (Transferrin‐⁵⁹Fe³⁺) and NTBI, whether presented as ⁵⁹Fe²⁺‐citrate or ⁵⁹Fe³⁺‐citrate; reductase‐independent ⁵⁹Fe²⁺ uptake was the most efficient uptake pathway of the three. Kinetic analysis of Zn²⁺ inhibition of Fe²⁺ uptake indicated that DMT1 plays only a minor role in the uptake of NTBI. In contrast, localization and knockdown data indicate that Zip8 makes a major contribution. Data suggest also that cell accumulation of ⁵⁹Fe from TBI relies at least in part on an endocytosis‐independent pathway. These data suggest that Zip8 and Steap2 play a major role in iron accumulation from NTBI and TBI by hippocampal neurons.

     

Non-Transferrin-Bound Iron (NTBI) Uptake by T Lymphocytes: Evidence for the Selective Acquisition of Oligomeric Ferric Citrate Species

Iron is an essential nutrient in several biological processes such as oxygen transport, DNA replication and erythropoiesis. Plasma iron normally circulates bound to transferrin. In iron overload disorders, however, iron concentrations exceed transferrin binding capacity and iron appears complexed with low molecular weight molecules, known as non-transferrin-bound iron (NTBI). NTBI is responsible for the toxicity associated with iron-overload pathologies but the mechanisms leading to NTBI uptake are not fully understood. Here we show for the first time that T lymphocytes are able to take up and accumulate NTBI in a manner that resembles that of hepatocytes. Moreover, we show that both hepatocytes and T lymphocytes take up the oligomeric Fe3Cit3 preferentially to other iron-citrate species, suggesting the existence of a selective NTBI carrier. These results provide a tool for the identification of the still elusive ferric-citrate cellular carrier and may also open a new pathway towards the design of more efficient iron chelators for the treatment of iron overload disorders.     

Non-transferrin bound iron measurement is influenced by chelator concentration

Release of non-protein bound iron plays an important role in the toxicity inflicted by chemotherapy in cancer patients. Since large variations have been described for different methods measuring non-transferrin bound iron (NTBI), we aimed to obtain more accurate values. After binding to the chelator nitrilotriacetic acid disodium salt (NTA) and ultrafiltration, the NTBI can be measured spectrophotometrically by the addition of thioglycolic acid (TGA) and baptophenanthroline disulfonic acid (BPT). Results demonstrated that NTBI values increased with NTA concentration. In samples incubated with 80 mM NTA, >5-fold higher NTBI values were found compared to using 10 mM NTA. Optimal concentration of NTA was established by additions of iron to serum with known latent iron-binding capacity (LIBC). Iron addition curves showed that NTBI could be measured starting from the LIBC of the serum with optimal yield after incubation with 4 mM NTA in 5 mM Tris-HCl pH 6.5, with 3 mM TGA and 6.2 mM BPT for the colour reaction. The results showed excellent correlation with 195 samples measured also by HPLC. For the spectrophotometric method, significantly higher NTBI values were measured in patient samples with maximal iron saturation compared to patients with lower iron saturation.     

Ascorbyl free radical reflects catalytically active iron after intravenous iron saccharate injection

Iron release from intravenous iron formulations can increase both non-transferrin-bound iron (NTBI) and oxidative stress. However, data showing a direct association between these parameters are sparse. The aim of this study was to adapt a recently published electron spin resonance (ESR) method to measure NTBI after iron injection and further to investigate its correlation to levels of oxidative stress markers. Twenty chronic hemodialysis patients were enrolled. NTBI and markers of oxidative stress, ascorbyl free radical (AFR), oxidized LDL, protein carbonyl, total antioxidant capacity, and myeloperoxidase, were measured in blood samples collected before and after intravenous injection of 100 mg iron saccharate. NTBI and all analyzed oxidative stress markers were increased 10 min after iron injection. Specifically, NTBI rose by 375% and AFR by 40%. Significant increases in these parameters were still seen 60 min after the injection. The changes in NTBI and AFR were closely correlated. The close correlation between intravascular release of NTBI and increase in plasma AFR after iv iron injection, as well as the increase in all measured oxidative stress markers, suggests that the iron measured was catalytically active. The ESR method was sufficiently sensitive and robust to measure NTBI also in human plasma.     

Nontransferrin-bound serum iron in thalassemia and sickle cell patients

Nontransferrin-bound iron (NTBI) was separated from transferrin bound iron (TBI) by DEAE-Sephadex-CDS filtration. TBI is eluted with Tris-NaCl buffer, NTBI that is retained on the column is eluted with citric acid. NTBI was identified in serum from thalassemia and sickle cell patients. Normal serum contained less than 6% NTBI as compared with 15-18% in patient's sera. NTBI levels were decreased significantly after 8 hr chelation with deferoxamine (DFO).     

Non-transferrin bound iron,cytokine activation and intracellular reactive oxygen species generation in hemodialysis patients receiving intravenous iron dextran or iron sucrose

Intravenous (IV) iron supplementation is widely used to support erythropoeisis in hemodialysis patients. IV iron products are associated with oxidative stress that has been measured principally by circulating biomarkers such as products of lipid peroxidation. The pro-oxidant effects of IV iron are presumed to be due at least in part, by free or non-transferrin bound iron (NTBI). However, the effects of IV iron on intracellular redox status and downstream effectors is not known. This prospective, crossover study compared cytokine activation, reactive oxygen species generation and oxidative stress after single IV doses of iron sucrose and iron dextran. This was a prospective, open-label, crossover study. Ten patients with end-stage renal disease (ESRD) on hemodialysis and four age and sex-matched healthy were assigned to receive 100 mg of each IV iron product over 5 min in random sequence with a 2 week washout between products. Subjects were fasted and fed a low iron diet in the General Clinical Research Center at the University of New Mexico. Serum and plasma samples for IL-1, IL-6, TNF-α and IL-10 and NTBI were obtained at baseline, 60 and 240 min after iron infusion. Peripheral blood mononuclear cells (PBMC) were isolated at the same time points and stained with fluorescent probes to identify intracellular reactive oxygen species and mitochondrial membrane potential (Δψm) by flow cytometry. Lipid peroxidation was assessed by plasma F₂ isoprostane concentration. Mean ± SEM maximum serum NTBI values were significantly higher among patients receiving IS compared to ID (2.59 ± 0.31 and 1.0 ± 0.36 μM, respectively, P = 0.005 IS vs. ID) Mean ± SEM NTBI area under the serum concentration–time curve (AUC) was 3-fold higher after IS versus ID (202 ± 53 vs. 74 ± 23 μM*min/l, P = 0.04) in ESRD patients, indicating increased exposure to NTBI. IV iron administration was associated with increased pro-inflammatory cytokines. Serum IL-6 concentrations increased most profoundly, with a 2.6 and 2.1 fold increase from baseline in ESRD patients given IS and ID, respectively (P < 0.05 compared to baseline). In healthy controls, serum IL-6 was undetectable at baseline and after IV iron administration. Most ESRD patients had increased intracellular ROS generation, however, there was no difference between ID and IS. Only one healthy control had increased ROS generation post IV iron. All healthy controls experienced a loss of Δψm (100% with IS and 50% with ID). ESRD patients also had loss of Δψm with a nadir at 240 min. IS administration was associated with higher maximum serum NTBI concentrations compared to ID, however, the both compounds produced similar ROS generation and cytokine activation that was more pronounced among ESRD patients. The effect of IV iron-induced ROS production on pivotal signaling pathways needs to be explored.     

Heart Rate Variability as an Alternative Indicator for Identifying Cardiac Iron Status in Non-Transfusion Dependent Thalassemia Patients

Background

Iron-overload cardiomyopathy is a major cause of death in thalassemia patients due to the lack of an early detection strategy. Although cardiac magnetic resonance (CMR) T2* is used for early detection of cardiac iron accumulation, its availability is limited. Heart rate variability (HRV) has been used to evaluate cardiac autonomic function and found to be depressed in thalassemia. However, its direct correlation with cardiac iron accumulation has never been investigated. We investigated whether HRV can be used as an alternative indicator for early identification of cardiac iron deposition in thalassemia patients.

Methods

Ninety-nine non-transfusion dependent thalassemia patients (23.00 (17.00, 32.75) years, 35 male) were enrolled. The correlation between HRV recorded using 24-hour Holter monitoring and non-transferrin bound iron (NTBI), hemoglobin (Hb), serum ferritin, LV ejection fraction (LVEF), and CMR-T2* were determined.

Results

The median NTBI value was 3.15 (1.11, 6.59) μM. Both time and frequency domains of HRV showed a significant correlation with the NTBI level, supporting HRV as a marker of iron overload. Moreover, the LF/HF ratio showed a significant correlation with CMR-T2* with the receiver operating characteristic (ROC) curve of 0.684±0.063, suggesting that it could represent the cardiac iron deposit in thalassemia patients. HRV was also significantly correlated with serum ferritin and Hb.

Conclusions

This novel finding regarding the correlation between HRV and CMR-T2* indicates that HRV could be a potential marker in identifying early cardiac iron deposition prior to the development of LV dysfunction, and may be used as an alternative to CMR-T2* for screening cardiac iron status in thalassemia patients.     

Quantification of non-transferrin-bound iron in the presence of unsaturated transferrin.

Non-transferrin-bound iron (NTBI) has been reported to be associated with several clinical states such as thalassemia, hemochromatosis, and in patients receiving chemotherapy. We have investigated a number of ligands as potential alternatives to nitrilotriacetic acid (NTA) to capture NTBI without chelating transferrin- or ferritin-bound iron in plasma. We have established, however, that NTA is the optimal ligand to chelate the different forms of NTBI present in sera and can be adopted for utilization in the NTBI assay. NTA (80 mM) removes all forms of NTBI, while only mobilizing a small fraction of the iron bound to both transferrin and ferritin. We have compared three different detection systems for the quantification of NTA-chelated NTBI: the established HPLC-based method, a simple colorimetric method, and a method based on inductive conductiometric plasma spectroscopy. The sensitivity and reproductibility of the colorimetric method were acceptable compared with the other two methods and would be more convenient as a routine laboratory screening assay for NTBI. However, the limitations of this method are such that it can only be utilized in situations where desferrioxamine is not used and when transferrin saturation levels are close to 100%. Only the HPLC-based method is applicable for patients receiving (desferrioxamine) chelation therapy. In some diseases such as hemochromatosis, transferrin may be incompletely saturated. In such cases, to avoid in vitro donation of iron onto the vacant sites of transferrin, sodium-tris-carbonatocobaltate(III) can be added to block the free iron binding sites on transferrin. If this step is not taken, there may be an underestimation of NTBI values.     

Effects of the iron chelator deferiprone and the T-type calcium channel blocker efonidipine on cardiac function and Ca2+ regulation in iron-overloaded thalassemic mice

Although disturbance of cardiac Ca²⁺ regulation is involved in the pathophysiology of iron-overload cardiomyopathy, the obvious mechanisms involved in the dysregulation of iron-induced cardiac Ca²⁺ are unclear. Moreover, the roles of the iron chelator deferiprone and the T-type calcium channel blocker efonidipine on cardiac intracellular Ca²⁺ transients and Ca²⁺ regulatory proteins in thalassemic mice are still unknown. We tested the hypothesis that treatment with either deferiprone or efonidipine attenuated cardiac Ca²⁺ dysregulation and led to improved left ventricular (LV) function in iron-overloaded thalassemic mice. Wild-type (WT) mice and β-thalassemic (HT) mice were fed with either a normal diet (ND) or a high iron-diet (FE) for 90 days. Then, the FE-fed mice were treated with either deferiprone (75 mg/kg/day) or efonidipine (4 mg/kg/day) for 30 days. ND-fed HT mice had an increase in T-type calcium channels (TTCC) and an increased level of sarcoplasmic reticulum Ca²⁺-ATPase (SERCA), compared with ND-fed WT mice. Chronic iron feeding led to an increase in TTCC and expression of SERCA proteins in FE-fed WT mice. Moreover, chronic iron overload led to increased plasma non-transferrin bound iron (NTBI) and cardiac iron deposition, impaired cardiac intracellular Ca²⁺ transients including decreased intracellular Ca²⁺ transient amplitude, rising rate and decay rate, as well as impaired LV function as indicated by a decreased %LV ejection fraction (%LVEF) in both WT and HT mice. Our findings showed that treatment with either deferiprone (DFP) or efonidipine (EFO) showed similar benefits in reducing plasma NTBI and cardiac iron deposition, and improving %LVEF from 84.3 (WT) to 89.3 (DFP) and 89.2 (EFO) treatment; and from 84.2 (HT) to 88.8 (DFP) and 89.5 (EFO) treatment, however there was no improvement in the regulation of cardiac Ca²⁺ in iron-overloaded thalassemic mice. These findings provide the understanding of the effects of these drugs on the iron-overloaded heart in thalassemic mice and suggest that an alternative intervention that could improve calcium regulation under this condition is needed to improve the therapeutic outcome. Moreover, whether the benefits of the TTCC blocker is via its inhibition of the TTCC alone or together with its ability to chelate iron are still unclear and need further investigation.     

Results of an international round robin for the quantification of serum non-transferrin-bound iron: Need for defining standardization and a clinically relevant isoform

Non-transferrin-bound iron (NTBI) appears in the circulation of patients with iron overload. Various methods to measure NTBI were comparatively assessed as part of an international interlaboratory study. Six laboratories participated in the study, using methods based on iron mobilization and detection with iron chelators or on reactivity with bleomycin. Serum samples of 12 patients with hereditary (n=11) and secondary (n=1) hemochromatosis were measured during a 3-day analysis using 4 determinations per sample per day, making a total of 144 measurements per laboratory. Bland-Altman plots for repeated measurements are presented. The methods differed widely in mean serum NTBI level (range 0.12-4.32mumol/L), between-sample variation (SD range 0.20-2.13mumol/L and CV range 49.3-391.3%), and within-sample variation (SD range 0.02-0.45mumol/L and CV range 4.4-193.2%). The results obtained with methods based on chelators correlated significantly (R(2) range 0.86-0.99). On the other hand, NTBI values obtained by the various methods related differently from those of serum transferrin saturation (TS) when expressed in terms of both regression coefficients and NTBI levels at TS of 50%. Recent studies underscore the clinical relevance of NTBI in the management of iron-overloaded patients. However, before measurement of NTBI can be introduced into clinical practice, there is a need for more reproducible protocols as well as information on which method best represents the pathophysiological phenomenon and is most pertinent for diagnostic and therapeutic purposes.     

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.     

Nontransferrin-bound Iron in Serum of Patients Receiving Bone Marrow Transplants

Nontransferrin-bound iron (NTBI) and other parameters of iron status were measured in 40 patients undergoing bone marrow transplantation (BMT) prior to conditioning therapy (between day −10 and −7), at the time of BMT (day 0), and 2 weeks later (day +14). Serum iron and transferrin saturation values were normal before conditioning therapy. At day 0 serum iron values were high and median transferrin saturation was 98% (changes in the values of both serum iron and transferrin saturation, p < .0001). Transferrin saturation values were still elevated 2 weeks posttransplant (day +14 vs. baseline values, p = .0001). Starting at low NTBI levels pretransplant (median 0.4 , range 0–4.2 , controls: ≤ 0.4 ), all patients revealed high levels on day 0 (median 4.0 , range 1.9–6.9 , p < .0001) and 2 weeks posttransplant (median 2.7 , range 0–6.2 , p < .0001). These observations indicate that the plasma iron pool in patients undergoing BMT increases to a level at which the normal ability to sequestrate iron becomes exhausted and considerable amounts of NTBI appear in serum. This “free” form of iron can mediate the production of reactive oxygen species and may cause organ toxicity in the early posttransplantation period. © 1997 Elsevier Science Inc.