Non-transferrin-bound Iron Levels Research Articles

Ferrophilic Characteristics ofVibrio vulnificusand Potential Usefulness of Iron Chelation Therapy

We determined the ferrophilic characteristics of Vibrio vulnificus to evaluate the potential usefulness of iron chelation therapy for the prevention of V. vulnificus infection. Readily available non-transferrin-bound iron (NTBI) is required for the initiation of V. vulnificus growth under in vitro iron-limited conditions and human ex vivo conditions. NTBI aided efficient transferrin-bound iron (TBI) use by V. vulnificus, and the vulnibactin-mediated iron-uptake system was expressed after bacterial growth had been started by NTBI. V. vulnificus required higher NTBI levels for the initiation of growth, produced siderophores at lower levels, and used TBI less efficiently than other bacteria. In addition, the growth of V. vulnificus was inhibited by deferiprone, a clinically available iron chelator. These results show that V. vulnificus is a ferrophilic bacterium that requires higher NTBI levels than other pathogens and that iron chelation therapy might be an effective means of preventing the in vivo growth of V. vulnificus in susceptible patients.

Non-Transferrin-Bound Iron and Oxidative Stress Are Related to the Degree of Erythropoiesis in Patients with Thalassemia Intermedia.

There is strong evidence that reactive oxygen species (ROS) are involved in the pathogenesis of thalassaemias. It has been shown that ROS are generated in increased amounts in thalassemic erythrocytes following the precipitation of excess unmatched globin chains and the deposition of non-heme iron and hemichromes. Continuous ROS production in thalassemic individuals may alter their overall redox status and cause tissue damage. Reduction in the levels of vitamin C, vitamin E, and carotenoids has been reported in beta-thalassemic patients receiving transfusion therapy. In this study we investigated the oxidative stress in relation to the degree of erythropoiesis in patients with beta-thalassemia Intermedia (TI). Forty patients with TI were included in the study. Sixteen patients had mild anemia and mild clinical phenotype. The remaining 24 patients had more severe phenotype, but rare transfusions had been used in only 8 of them. Non-transferrin-bound iron (NTBI) levels were determined using graphite furnace atomic absorption spectrometry. Lipid peroxidation expressed as malonyldialdehyde (MDA) concentration was measured by reverse-phase HPLC with fluorimetric detection. The erythroid marrow activity was estimated by measuring soluble transferrin receptors (sTfR) levels with a turbidimetric technique. The main results of the study showed thatNTBI and MDA levels were increased (normal controls <0.05micromol/L and <0.65 micromol/L respectively) in 32/40 patients, while sTfR was found 4- to 20-fold higher than normal in all patients,NTBI, MDA and sTFR levels were significantly higher in patients with the more severe phenotype compared with patients with the milder phenotype (p<0.01),NTBI correlated positively: with MDA (rho=0.502, p<0.003), with sTfR (rho=0.371, p<0.02) and Hb F (rho=0.464, p<0.005), while no correlation was found between NTBI and Hb levels (p>0.278) andMDA was correlated significantly with Hb F (rho=0.464, p<0.005), while the correlation with sTfR levels was poor (rho=0.313, p<0.05).Patients with thalassemia intermedia have increased MDA levels, suggestive of imbalanced oxidant/antioxidant equilibrium. This phenomenon seems to be related to altered iron homoestasis, especially with the increased levels of NTBI. NTBI induces ROS production mainly through the Fenton reaction. NTBI levels are affected by the severity of tissue hypoxia, as these were expressed by their positive correlations with levels of sTfR, MDA and Hb F. The following hypothesis may be explain our findings; increased Hb F leads to tissue hypoxia, which induces further erythropoetic activity, as this is expressed by increased sTfR levels. Increased erythropoetic activity, possibly through a negative effect on hepcidin production (Kattamis et al, Haematologica 91: 809–12, 2006) results in increased NTBI levels, which lead to increased ROS production and MDA levels. Further studies are required to validate this hypothesis that altered iron cycle contributes to the oxidant/antioxidant imbalance observed in thalassemia intermedia.

Identification of a New Mutation in the 5′-UTR of Hepcidin Gene in beta-Thalassemia Major (TM) Patients.

Hepcidin is a peptide hormone produced in the liver that acts as negative regulator of iron absorption from the enterocytes and of iron release from macrophages. Iron overload and inflammation up-regulate hepcidin synthesis, while anaemia and hypoxia suppress hepcidin expression. Thalassaemia Major (TM) is a hereditary haemolytic anaemia requiring long-life blood transfusions treatment with consequent iron overload. In β-thalassemias is a disorder in which hepcidin is regulated by opposing influences of ineffective erythropoiesis and concomitant iron overload. In order to get further insights on iron regulation in thalassemias, we screened hepcidin and HFE genes in fourty-three TM regularly transfused patients and sixty control subjects. Blood from TM was taken at least 48 hours after chelation therapy and just before blood transfusion. DNA was prepared from peripheral blood, according to standard protocols. Hepcidin and HFE sequences were amplified with PCR using specific primers and PCR products were sequenced, after purification, in a automatic sequencer. Moreover in all patients serum pro-hepcidin was evaluated by ELISA competitive binding assay (DRG,Germany); iron status was evaluated by serum ferritin (SF), percentage of transferrin saturation (TS) by standard procedures and non transferrin bound iron (NTBI) in serum by HPLC after nitrilotriacetic acid (NTA) chelation. Serum IL-6 as inflammation marker was measured by lateral flow immunoassay (Milenia QuickLine, Germany). Molecular analysis detected an undescribed G→T mutation at position +23 of the 5′-untranslated region in two unrelated TM patients; no mutations were found in control subjects. The probands have been regularly transfused since the age of 1 year, receiving 2–3 units of packed red cells and treated with Deferoxamine 40 mg/Kg/day 6 days/week. The first patient, wild type for HFE mutation, was a 29-years-old compound heterozygous IVS II-745/IVS I-110 man. The SF was 1052 ng/ml, TS 94% and NTBI 1.77 μM. Serum pro-hepcidin value was in normal range (213 ng/ml). The proband's father was heterozygous for the same hepcidin mutation and showed signs of mild iron overload (SF 491 ng/ml and NTBI 0.50 μM). The second patient was a 26-years-old homozygous β039 man with high levels of SF (4346 ng/ml), TS (169%) and NTBI (2.10 μM), while serum pro-hepcidin was 269 ng/ml. HFE analysis revealed a homozygous genotype for H63D mutation. The patient's mother was heterozygous for hepcidin and H63D mutation and showed mild iron overload (SF 500 ng/ml; NTBI 0.22 μM) whereas the father, heterozygous only for H63D, had normal iron status. According to recent findings (Bridle et al, 2003) our results indicate that hepcidin mutation in association with H63D synergizes the effect on iron homeostasis and it could be responsible for the development of marked iron overload poorly responsive to chelation therapy in β-thalassemia patients.

Common Presence of Non–Transferrin-Bound Iron Among Patients With Type 2 Diabetes

OBJECTIVE—Recently, we reported increased cardiovascular disease mortality among supplemental vitamin C users with type 2 diabetes in a prospective cohort study. Because vitamin C may cause oxidative stress in the presence of redox active iron, we hypothesized that non–transferrin-bound iron (NTBI), a form of iron susceptible to redox activity, may be present in patients with type 2 diabetes. RESEARCH DESIGN AND METHODS—We measured serum NTBI levels using high-performance liquid chromatography in 48 patients with known diabetes (at least 5 years duration since diagnosis), 49 patients with newly diagnosed diabetes, and 47 healthy control subjects (frequency matched on age and sex). RESULTS—NTBI was commonly present in diabetes: 59% in newly diagnosed diabetes and 92% in advanced diabetes. Mean NTBI values varied significantly between the three groups, with the highest values being observed in patients with known diabetes and the lowest in the control subjects (0.62 ± 0.43 vs. 0.24 ± 0.29 vs. 0.04 ± 0.13 μmol/l Fe). Serum total iron or percent transferrin saturation were very similar among the three groups, yet NTBI was strongly associated with serum total iron (r = 0.74, P &amp;lt; 0.01) and percent transferrin saturation (r = 0.70, P &amp;lt; 0.01) among the patients with known diabetes. CONCLUSIONS—Consistent with our hypothesis, these data demonstrate the common existence of NTBI in type 2 diabetic patients with a strong gradient with severity. Prospective cohort studies are required to clarify the clinical relevance of increased NTBI levels.

Common Presence of Non-Transferrin-Bound Iron Among Patients With Type 2 Diabetes

Recently, we reported increased cardiovascular disease mortality among supplemental vitamin C users with type 2 diabetes in a prospective cohort study. Because vitamin C may cause oxidative stress in the presence of redox active iron, we hypothesized that non-transferrin-bound iron (NTBI), a form of iron susceptible to redox activity, may be present in patients with type 2 diabetes. We measured serum NTBI levels using high-performance liquid chromatography in 48 patients with known diabetes (at least 5 years duration since diagnosis), 49 patients with newly diagnosed diabetes, and 47 healthy control subjects (frequency matched on age and sex). NTBI was commonly present in diabetes: 59% in newly diagnosed diabetes and 92% in advanced diabetes. Mean NTBI values varied significantly between the three groups, with the highest values being observed in patients with known diabetes and the lowest in the control subjects (0.62 +/- 0.43 vs. 0.24 +/- 0.29 vs. 0.04 +/- 0.13 micromol/l Fe). Serum total iron or percent transferrin saturation were very similar among the three groups, yet NTBI was strongly associated with serum total iron (r = 0.74, P < 0.01) and percent transferrin saturation (r = 0.70, P < 0.01) among the patients with known diabetes. Consistent with our hypothesis, these data demonstrate the common existence of NTBI in type 2 diabetic patients with a strong gradient with severity. Prospective cohort studies are required to clarify the clinical relevance of increased NTBI levels.

Issue Highlights

HomeCirculationVol. 113, No. 16Issue Highlights Free AccessIn BriefPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessIn BriefPDF/EPUBIssue Highlights Originally published25 Apr 2006https://doi.org/10.1161/circ.113.16.1921Circulation. 2006;113:1921NON–TRANSFERRIN-BOUND IRON AND RISK OF CORONARY HEART DISEASE IN POSTMENOPAUSAL WOMEN, by van der A et al.The parallel rise in both body iron stores and vascular risk in women after menopause led to the ‘iron overload‘ theory of coronary heart disease (CHD). Epidemiological studies relating serum ferritin levels to CHD risk have yielded inconsistent results, however. In this issue of Circulation, van der A and colleagues evaluated a community-based sample of postmenopausal women to test the hypothesis that a labile iron component associated with non--transferrin-bound iron (NTBI) might be more suitable for relating to CHD risk. The investigators noted that women in the highest NTBI tertile had a statistically significant 53% lower risk of acute myocardial infarction and a nonsignificant 16% lower risk of CHD compared with those in the lowest tertile in multivariable analyses. Similar results were obtained for serum iron, transferrin saturation, and serum ferritin. These observations do not support the presence of an excess risk of CHD or myocardial infarction in postmenopausal women with relatively higher levels of NTBI or other measures of iron overload. See p 1942.STEM CELL MOBILIZATION INDUCED BY SUBCUTANEOUS GRANULOCYTE-COLONY STIMULATING FACTOR TO IMPROVE CARDIAC REGENERATION AFTER ACUTE ST-ELEVATION MYOCARDIAL INFARCTION: RESULT OF THE DOUBLE-BLIND, RANDOMIZED, PLACEBO-CONTROLLED STEM CELLS IN MYOCARDIAL INFACTION (STEMMI) TRIAL, by Ripa et al.Percutaneous coronary revascularization procedures restore coronary flow quickly in patients with acute ST-elevation myocardial infarction, and yet there is often some degree of myocardial necrosis resulting in left ventricular dysfunction. To prevent adverse ventricular remodeling, efforts have been devoted to determining the therapeutic efficacy of bone-marrow–derived stem cells in preserving ventricular function. These cells have been isolated ex vivo and infused into the coronary artery or have been stimulated to exit the bone marrow to home to sites of myocardial injury by the administration of granulocyte-colony stimulating factor (G-CSF). Early open-label studies have demonstrated the safety and potential efficacy of G-CSF. In this issue of Circulation, Ripa et al present data from the STEM cells in Myocardial Infarction (STEMMI) Trial. This is the first report of a randomized, double-blind, placebo-controlled trial of G-CSF in patients with ST-elevation myocardial infarction and successful percutaneous revascularization. See p 1983.MACROPHAGE METALLOELASTASE ACCELERATES THE PROGRESSION OF ATHEROSCLEROSIS IN TRANSGENIC RABBITS, by Liang et al.In this issue of Circulation, Liang et al used a novel approach involving the generation of transgenic rabbits to study the role of the macrophage metalloelastase (matrix metalloproteinase [MMP]-12) in atherosclerosis. Their results indicate that MMP-12 causes media destruction and pseudoaneurysm formation and accelerates plaque growth in an animal model that more closely resembles atherosclerosis in humans. This transgenic rabbit model may offer new insights into the roles of MMPs in extracellular matrix remodelling and cellular migration in atherosclerotic lesion formation that may challenge some prior conclusions based on mouse models. See p 1993.Visit http://circ.ahajournals.org:Cardiology Patient PageSyncope. See p e715.Images in Cardiovascular MedicineNatural Tissue Engineering Inside a Ventricular Septum Defect Occluder. See p e718. Download figureDownload PowerPointTransition From Left Ventricular Hypertrophy to Massive Fibrosis in the Cardiac Variant of Fabry Disease. See p e720.External Carotid Artery--Internal Jugular Vein Fistula: A Complication of Internal Jugular Cannulation. See p e722.CorrespondenceSee p e724. Previous Back to top Next FiguresReferencesRelatedDetails April 25, 2006Vol 113, Issue 16 Advertisement Article InformationMetrics https://doi.org/10.1161/circ.113.16.1921 Originally publishedApril 25, 2006 PDF download Advertisement

Iron sucrose augments homocysteine-induced endothelial dysfunction in normal subjects

Intravenous iron is commonly used in conjunction with erythropoietic agents to treat anemia in patients with chronic kidney disease. Iron has been proposed to promote oxidative stress and endothelial dysfunction in vascular tissues. We studied the acute effects of intravenous iron sucrose on homocysteine-induced endothelial dysfunction in the brachial artery of normal human subjects. In all, 40 healthy subjects received intravenous iron sucrose 100 mg or placebo over 30 min immediately before ingestion of 100 mg/kg of oral methionine in a double-blind, randomized study. Flow- and nitroglycerin-mediated dilation in the brachial artery, serum markers of iron stores, and homocysteine and nitrotyrosine levels were measured before and after study drug administration. Intravenous iron significantly increased transferrin saturation and non-transferrin-bound iron (NTBI) when compared with placebo. Flow-mediated dilation significantly decreased from baseline 1 h after administration of iron sucrose when compared with placebo (from 6.66+/-0.47 to 1.93+/-0.35% after iron sucrose vs from 6.00+/-0.40 to 5.61+/-0.46% after placebo, P<0.001), but did not differ between groups at 4 h (1.10+/-0.39 vs 1.33+/-0.51%). Nitroglycerin-mediated vasodilation, and homocysteine and 3-nitrotyrosine levels did not differ after administration of iron sucrose and placebo. Intravenous administration of iron sucrose in the setting of transient hyperhomocysteinemia induced by methionine ingestion significantly increased transferrin saturation and plasma levels of NTBI and significantly attenuated flow-mediated dilation in the brachial artery when compared with placebo. This potential mechanistic link between intravenous iron and endothelial dysfunction warrants further study of cardiovascular effects of intravenous iron in anemic chronic kidney disease populations.

Endothelial activation and induction of monocyte adhesion by nontransferrin‐bound iron present in human sera

Nontransferrin-bound iron (NTBI) has been detected in iron overload diseases. This form of iron may exert pro-oxidant effects and modulate cellular function and inflammatory response. The present study has aimed to investigate the effects of serum NTBI on monocyte adherence to endothelium. Measured by a recently developed high-throughput fluorescence-based assay, serum NTBI was found to be higher in both homozygotes of HFE C282Y mutation of hereditary hemochromatosis (7.9+/-0.6 microM, n=9, P<0.001) and heterozygotes (4.0+/-0.5 microM, n=8, P<0.001), compared with controls (1.6+/-0.2 microM, n=21). The effects of these sera on monocyte adhesion and endothelial activation were examined. Adhesion of normal human monocytes to C282Y homozygote- and heterozygote-serum-treated human umbilical vein endothelial cells was higher (25.0+/-0.9 and 22.1+/-0.7%, respectively) compared with controls (17.6+/-0.5%, both P<0.001). For the three groups combined, the expression of adhesion molecules, ICAM-1, VCAM-1, and E-selectin, was positively correlated to NTBI levels but not to the inflammatory marker C-reactive protein. Furthermore, accumulation of intracellular labile iron and oxidative radicals within the cells due to NTBI was evidenced. Finally, counteraction of NTBI-induced endothelial activation was observed using iron chelators. These findings therefore identify a physiological function of NTBI in monocyte-endothelial interactions that may also contribute to the development of atherosclerosis and neurodegenerative diseases.

Non‐Transferrin‐Bound Iron during Blood Transfusion Cycles in β‐Thalassemia Major

Serum non-transferrin-bound iron (NTBI) levels assessed at arbitrary time points during transfusion cycles may not be representative if NTBI is undergoing significant changes during transfusion cycles. In 15 patients with beta-thalassemia major (age: 21 +/- 6 years, liver iron concentration: 2200 +/- 1200 microg/g-liver), NTBI and other hematologic parameters (transferrin saturation, transferrin receptor) were measured weekly. The largest variation of NTBI levels between individual patients was observed at midcycle. For long-term monitoring of NTBI levels, a particular time point relative to the last blood transfusion should be selected for blood drawing.

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 0.12–4.32 μmol/L), between-sample variation (SD range 0.20–2.13 μmol/L and CV range 49.3–391.3%), and within-sample variation (SD range 0.02–0.45 μmol/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.

Decreased Serum Pro-Hepcidin in Thalassaemia Intermedia; a Mechanism for Increased Iron Absortion Mediated Though Ineffective Erthropoiesis and NTBI?.

Background: Pro-hepcidin is 10 kDa peptide localized at the basolateral membrane of hepatocytes which is also present in plasma and is believed to be a hepcidin precursor (Kulaksis et al, Gut 2004;53). Experimental hepatic iron loading in mice up-regulates hepcidin mRNA while iron deficiency has the reverse effect (Pigeon et al, JBC 2001;276). Paradoxically mice with a thalassaemia intermedia phenotype had reduced liver hepcidin mRNA. Furthermore, presentation of non-transferrin bound iron (NTBI) to human liver cells (Nemeth, Blood 2003; 101)(Rafique, 2004 in preparation) decreased hepcidin mRNA. Patients and methods: 3 groups of patients were compared (thalassaemia major (TM), thalassaemia intermedia (TI) and sickle cell anaemia (HbSS)) to examine if variations in pro-hepcidin expression could account for the differences in iron loading. Pro-hepcidin measurements were performed at 1:1 dilutions in duplicate with a Pro-hepcidin ELISA (DRG diagnostics) using a previously published method (Kulakis, 2004). Iron status was evaluted by liver CMR , serum ferritin and NTBI; C reactive protein (CRP) was measured to exclude inflammation. All patients with a raised creatinine were excluded from the analysis. Results: TI patients (n=20) had a significantly lower serum pro-hepcidin (77 ± 40ng/ml) ( p<0.001) compared to HbSS (n=26) (111 ± 53 ng/ml), TM (n=27) (107 ± 44.5ng/ml) or control subjects (n=9) (119 ± 45ng/ml). The difference in serum pro-hepicidin between TI and HbSS persisted when patients were matched for serum ferritin (p = 0.02) or liver iron (p < 0.001). NTBI was significantly lower in HbSS (1.06 ± 0.27umol/l) than TI (2.49 ± 2.01 umol/l) even when patients were matched for serum ferritin (p <0.001) or for liver iron (p <0.05). Hb values were not significantly different between TI (8.6 g/dl) and HbSS patients (8.9g/dl). When all patients with a Hb <8g/dl were compared to those with a Hb > 10g/dl serum pro-hepcidin was not significantly different. No correlation between serum pro-hepcidin and liver iron, serum ferritin, CRP or Hb in any of the three groups was seen. Conclusion: Pro-hepcidin is significantly lower in TI than TM or HbSS patients and this cannot be accounted for by levels of anemia. We propose that NTBI, present early in TI secondary to high ineffective erythropoiesis, may lead to a similar paradoxical decrease in hepcidin synthesis reported in model systems and that this is reflected by reduced serum pro-hepcidin. Although levels of NTBI and liver iron did not differ significantly in TI and TM , transfusion and chelation therapy in TM may overide the NTBI mediated down regulation of pro-hepcidin. In HbSS, NTBI is lower than in TI or TM at matched levels of iron loading, resulting in less inhibition of pro-hepcidin synthesis and less duodenal iron absorption than in TI.

Nontransferrin-bound iron uptake by hepatocytes is increased in theHfeknockout mouse model of hereditary hemochromatosis

AbstractHereditary hemochromatosis (HH) is an iron-overload disorder caused by a C282Y mutation in theHFEgene. In HH, plasma nontransferrin-bound iron (NTBI) levels are increased and NTBI is bound mainly by citrate. The aim of this study was to examine the importance of NTBI in the pathogenesis of hepatic iron loading inHfeknockout mice. Plasma NTBI levels were increased 2.5-fold inHfeknockout mice compared with control mice. Total ferric citrate uptake by hepatocytes isolated fromHfeknockout mice (34.1 ± 2.8 pmol Fe/mg protein/min) increased by 2-fold compared with control mice (17.8 ± 2.7 pmol Fe/mg protein/min;P< .001; mean ± SEM; n = 7). Ferrous ion chelators, bathophenanthroline disulfonate, and 2′,2-bipyridine inhibited ferric citrate uptake by hepatocytes from both mouse types. Divalent metal ions inhibited ferric citrate uptake by hepatocytes, as did diferric transferrin. Divalent metal transporter 1 (DMT1) mRNA and protein expression was increased approximately 2-fold by hepatocytes fromHfeknockout mice. We conclude that NTBI uptake by hepatocytes fromHfeknockout mice contributed to hepatic iron loading. Ferric ion was reduced to ferrous ion and taken up by hepatocytes by a pathway shared with diferric transferrin. Inhibition of uptake by divalent metals and up-regulation of DMT1 expression suggested that NTBI uptake was mediated by DMT1.

Non-transferrin bound iron uptake by hepatocytes is increased in a Hfe knockout mouse model of hereditary hemochromatosis

Hereditary hemochromatosis (HH) is an iron-overload disorder caused by a C282Y mutation in the HFE gene. In HH, plasma nontransferrin-bound iron (NTBI) levels are increased and NTBI is bound mainly by citrate. The aim of this study was to examine the importance of NTBI in the pathogenesis of hepatic iron loading in Hfe knockout mice. Plasma NTBI levels were increased 2.5-fold in Hfe knockout mice compared with control mice. Total ferric citrate uptake by hepatocytes isolated from Hfe knockout mice (34.1 2.8 pmol Fe/mg protein/min) increased by 2-fold compared with control mice (17.8 2.7 pmol Fe/mg protein/min; P < .001; mean SEM; n 7). Ferrous ion chelators, bathophenanthroline disulfonate, and 2,2-bipyridine inhibited ferric citrate uptake by hepatocytes from both mouse types. Divalent metal ions inhibited ferric citrate uptake by hepatocytes, as did diferric transferrin. Divalent metal transporter 1 (DMT1) mRNA and protein expression was increased approximately 2-fold by hepatocytes from Hfe knockout mice. We conclude that NTBI uptake by hepatocytes from Hfe knockout mice contributed to hepatic iron loading. Ferric ion was reduced to ferrous ion and taken up by hepatocytes by a pathway shared with diferric transferrin. Inhibition of uptake by divalent metals and up-regulation of DMT1 expression suggested that NTBI uptake was mediated by DMT1. (Blood. 2004;104: 1519-1525)

Increased non-transferrin bound iron in plasma-depleted SAG-M red blood cell units.

Non-transferrin bound iron (NTBI) is associated with increased morbidity in a number of transfusion-dependent disease states such as the severe haemoglobinopathies. We hypothesized that this may be related to excess NTBI present in plasma-depleted red blood cell units that are free of clear haemolysis. The level of NTBI was determined using the bleomycin assay in samples from 20 stored plasma-depleted red cell units, at approximate 5-day intervals up to day 33 after donation. Forty units of fresh-frozen plasma (FFP) and 40 units of platelet concentrates were used as negative controls, and samples from 12 units of FFP were also serially assessed. Median [interquartile range (IQR)] NTBI was 0 microm (0-0.35) in samples taken from units 3-10 days after donation. Thereafter, the levels of NTBI increased, becoming significant (median 3.05; IQR: 0.05-6.7 microm) 17-22 days after donation. After 30 days, NTBI was detectable in all red cell units. NTBI was undetectable in platelet concentrates and FFP. Increased levels of NTBI become detectable 17-22 days after donation and increase further with storage time. This excess NTBI may promote bacterial infection in iron-loaded individuals.

Oxidative status and malondialdehyde in beta-thalassaemia patients.

In beta-thalassaemia syndromes, decreased or impaired biosynthesis of beta-globin leads to accumulation of unpaired alpha-globin chains. Moreover, the iron overload in beta-thalassaemia patients generates oxygen-free radicals and peroxidative tissue injury. The aim of this study was to detect and correlate iron overload parameters with the oxidative stress and the antioxidant capability in beta-thalassaemia patients. Serum iron, transferrin saturation, serum ferritin, nontransferrin-bound iron (NTBI), levels of serum free and total (free + bound) malondialdehyde (MDA) and total peroxyl radical-trapping antioxidant parameter (TRAP) were evaluated in 21 regularly transfused beta-thalassaemia major (TM) patients, 13 untransfused beta-thalassaemia intermedia (TI) patients and 17 healthy controls. Blood from the TM patients was drawn 48 h after the last desferoxamine (20-40 mg kg(-1)) infusion and just before transfusion. Free and total MDA and NTBI levels were higher in the TM patients than in the TI. In the TM patients the free MDA levels correlated positively with serum iron (r = +0.3, P = 0.0006), whereas the total MDA correlated positively with NTBI (r = +0.45, P = 0.037). However, a negative correlation was observed between TRAP and NTBI (r = -0.4, P = 0.0006). In the TI patients there was no significant correlation between free or total MDA and TRAP or NTBI. Our results confirm the peroxidative status generated by iron overload in thalassaemia patients and highlight the rapid formation of marked amounts of free MDA despite the chelation therapy in TM patients.

Sensitive method for nontransferrin-bound iron quantification by graphite furnace atomic absorption spectrometry

Objective: To establish a sensitive method for measuring nontransferrin-bound iron (NTBI) in serum samples using graphite furnace atomic absorption spectrometry (GFAAS). Design and methods: Nontransferrin-bound iron (NTBI) was chelated using nitrilotriacetic acid (NTA) and then ultrafiltered according to the method employed by Singh et al. [1]. Serum ultrafiltrates were diluted eightfold with distilled water. NTBI from the Fe-NTA complex present in the serum ultrafiltrate was measured using GFAAS. Results: Nontransferrin-bound iron (NTBI) and other parameters were measured in seven patients diagnosed with hereditary hemochromatosis by liver biopsy. Total serum iron, NTBI and transferrin saturation values (ranging from 87% to 90%) were elevated for three of the seven hemochromatosis patients tested before therapeutic phlebotomy. Six of the seven hemochromatosis patients had undergone phlebotomy and revealed normal total serum iron, NTBI and transferrin saturation values. Nine test subjects (not diagnosed with hemochromatosis) with abnormally high total serum iron and/or ferritin concentrations exhibited normal NTBI values (≤0.14 μmol/L to 0.29 μmol/L). The detection limit was 0.1 μmol/L for a 25 μL injection volume. Conclusions: The GFAAS method presented here provides a sensitive assay to quantitate NTBI in serum samples. The method developed is 4 to 5 times more sensitive than the only other GFAAS method [2] and more than an order of magnitude more sensitive than other colorimetric methods [1,3]. Improvement in sensitivity over the other GFAAS method [2] may be accounted for by differences in sample preparation between this method and that of Nielsen et al. [2]. Serum ultrafiltrates in this study were diluted eightfold with distilled water and mixed with a magnesium nitrate matrix modifier before GFAAS analysis. NTBI results obtained from this study indicate that the plasma iron pool in hemochromatosis patients awaiting phlebotomy increases to a level at which transferrin’s ability to bind iron becomes exhausted and elevated NTBI levels appear in the serum. NTBI can mediate the production of reactive oxygen species and may cause organ damage associated with iron overload.

Impaired plasma antioxidative defense and increased nontransferrin-bound iron during high-dose chemotherapy and radiochemotherapy preceding bone marrow transplantation

To analyze the effects of radiochemotherapy on the pro-oxidative/antioxidative balance in plasma, we measured the total radical antioxidant parameter of plasma (TRAP) and single plasma antioxidants (uric acid, sulfhydryl groups, α-tocopherol, ubiquinone-10/total coenzyme-Q10 ratio, ascorbate, and bilirubin) every 12 h during high-dose chemotherapy and radiochemotherapy preceding bone marrow transplantation (BMT). Nontransferrin-bound iron (NTBI) was monitored as a potential pro-oxidant. Plasma levels of polyunsaturated fatty acids (PUFA) were measured as substrates, and thiobarbituric acid–reactive substances (TBARS) were measured as products of lipid peroxidation. Allantoin was analyzed as the product of uric acid oxidation. Patients receiving busulfan, VP-16, and cyclophosphamide (BU/VP/CY) ( n = 8) were compared with those receiving total body irradiation in addition to VP-16 and cyclophosphamide (TBI/VP/CY) ( n = 8). TRAP values were within the normal range before therapy and decreased after BU/VP/CY by 37% ( p < .02) and after TBI/VP/CY by 39% ( p < .02). During TBI and after VP-16, a temporary increase in TRAP values occurred, which was not related to changes in individual antioxidants. In vitro experiments confirmed that VP-16 had an antioxidative effect. The concentration of uric acid declined in both groups and correlated with TRAP (BU/VP/CY: r = .80, p < .001; TBI/VP/CY: r = .84, p < .001). Levels of NTBI, which is normally not found in plasma, increased rapidly during conditioning therapy ( p < .02 in both groups) and correlated inversely with TRAP (weighted intraindividual Spearman rank correlation coefficient for both groups: NTBI and TRAP: r = −.59, p < .001) and PUFA (in the radiochemotherapy group: r = −.67, p < .001). Whereas PUFA declined ( p < .02 in both groups), TBARS increased ( p < .05 in both groups). Furthermore, an increase of allantoin and ubiquinone-10/total coenzyme-Q10 ratio in the BU/VP/CY group was found (allantoin: p < .02; ubiquinone-10/total coenzyme-Q10 ratio: p < .05). Antioxidants only partially recovered to baseline values until day 14 after BMT. Our findings indicate oxidative stress after high-dose radiochemotherapy and suggest a contribution of NTBI therein.

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 μmol/l, range 0–4.2 μmol/l, controls: ≤ 0.4 μmol/l), all patients revealed high levels on day 0 (median 4.0 μmol/l, range 1.9–6.9 μmol/l, p < .0001) and 2 weeks posttransplant (median 2.7 μmol/l, range 0–6.2 μmol/l, 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.

Non-transferrin-bound iron in long-term transfusion in children with congenital anemias

Non-transferrin-bound iron (NTBI), a potentially toxic compound, is increased in the serum of patients with iron overload and fully saturated transferrin. We found markedly elevated NTBI levels in 16 children (including nine with sickle cell disease and five with beta-thalassemia) with iron overload secondary to prolonged transfusion therapy. During iron chelation with subcutaneous desferrioxamine infusion, NTBI levels decreased to normal, but became elevated within 2 to 4 hours after discontinuation of desferrioxamine. NTBI causes hepatic and cardiac toxicity in experimental systems, but our patients lacked sufficient organ dysfunction for this association to be made. The use of continuous 24-hour chelation to maintain NTBI at low levels may prevent progressive iron toxicity in patients who first received chelation therapy at an older age or who already have evidence of cardiac damage.

Nontransferrin-bound serum iron in thalassemia and sickle cell patients

1. 1. 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. 2. 2. NTBI was identified in serum from thalassemia and sickle cell patients. Normal serum contained less than 6% NTBI as compared with 15–18% in patients' sera. 3. 3. NTBI levels were decreased significantly after 8 hr chelation with deferoxamine (DFO).