Iron-Chelating and Free-Radical Scavenging Activities of Microwave-Processed Green Tea in Iron Overload

Secondary iron overload is found in β-thalassemia (thal) patients because of increased dietary iron absorption and multiple blood transfusions. Excessive iron catalyzes free-radical generation, leading to oxidative damage and vital organ dysfunction. Non-transferrin-bound iron (NTBI) detected in thalassemic plasma is highly toxic and chelatable. Though used to treat iron overload, desferrioxamine (DFO) and deferiprone (L1) also have adverse effects. Green tea (GT) shows many pharmacological effects, particularly antioxidative and iron-chelating capacities. This study was performed to investigate the ability of GT extracts to reduce plasma NTBI concentration and oxidative stress in vitro. The Fe³⁺ was found to bind to GT crude extract and form a complex. Green tea crude extract time- and dose-dependently decreased plasma NTBI concentration and counteracted the increase of oxidative stress in both Fe ²⁺-EDTA-treated human plasma and erythrocytes. Green tea is a bifunctional natural product that could be relevant for management of iron overload and oxidative stress.     

Analytical evaluation of serum non–transferrin-bound iron and its relationships with oxidative stress and cardiac load in the general population

Excessive iron accumulation provokes toxic effects, especially in the cardiovascular system. Under iron overload, labile free non–transferrin-bound iron (NTBI) can induce cardiovascular damage with increased oxidative stress. However, the significance of NTBI in individuals without iron overload and overt cardiovascular disease has not been investigated. We aimed to examine the distribution of serum NTBI and its relationship with oxidative stress and cardiac load under physiological conditions in the general population.We enrolled individuals undergoing an annual health check-up and measured serum NTBI and derivatives of reactive oxygen metabolites (d-ROM), an oxidative stress marker. In addition, we evaluated serum levels of B-type natriuretic peptide (BNP) to examine cardiac load. We excluded patients with anemia, renal dysfunction, cancer, active inflammatory disease, or a history of cardiovascular disease.A total of 1244 individuals (57.8 ± 11.8 years) were enrolled, all of whom had detectable serum NTBI. d-ROM and BNP showed significant trends across NTBI quartiles. Multivariable regression analysis revealed that serum iron and low-density lipoprotein cholesterol were positively associated with NTBI but that age, d-ROM, and BNP showed an inverse association with this measure. In logistic regression analysis, NTBI was independently associated with a combination of higher levels of both d-ROM and BNP than the upper quartiles after adjustment for possible confounding factors.Serum NTBI concentration is detectable in the general population and shows significant inverse associations with oxidative stress and cardiac load. These findings indicate that serum NTBI in physiological conditions does not necessarily reflect increased oxidative stress, in contrast to the implications of higher levels in states of iron overload or pathological conditions.     

Zip14 (Slc39a14) mediates non-transferrin-bound iron uptake into cells

Zip14 is a member of the SLC39A zinc transporter family, which is involved in zinc uptake by cells. Up-regulation of Zip14 by IL-6 appears to contribute to the hepatic zinc accumulation and hypozincemia of inflammation. At least three members of the SLC39A family transport other trace elements, such as iron and manganese, in addition to zinc. We analyzed the capability of Zip14 to mediate non-transferrin-bound iron (NTBI) uptake by overexpressing mouse Zip14 in HEK 293H cells and Sf9 insect cells. Zip14 was found to localize to the plasma membrane, and its overexpression increased the uptake of both (65)Zn and (59)Fe. Addition of bathophenanthroline sulfonate, a cell-impermeant ferrous iron chelator, inhibited Zip14-mediated iron uptake from ferric citrate, suggesting that iron is taken up by HEK cells as Fe(2+). Iron uptake by HEK and Sf9 cells expressing Zip14 was inhibited by zinc. Suppression of endogenous Zip14 expression by using Zip14 siRNA reduced the uptake of both iron and zinc by AML12 mouse hepatocytes. Zip14 siRNA treatment also decreased metallothionein mRNA levels, suggesting that compensatory mechanisms were not sufficient to restore intracellular zinc. Collectively, these results indicate that Zip14 can mediate the uptake of zinc and NTBI into cells and that it may play a role in zinc and iron metabolism in hepatocytes, where this transporter is abundantly expressed. Because NTBI is commonly found in plasma of patients with hemochromatosis and transfusional iron overload, Zip14-mediated NTBI uptake may contribute to the hepatic iron loading that characterizes these diseases.     

Non-transferrin-bound iron and hepatic dysfunction in African dietary iron overload

BACKGROUND: Circulating iron is normally bound to transferrin. Non-transferrin-bound iron (NTBI) has been described in most forms of iron overload, but has not been studied in African dietary iron overload. This abnormal iron fraction is probably toxic, but this has not been demonstrated. METHODS: High-pressure liquid chromatography was used to assay serum NTBI in 25 black African subjects with iron overload documented by liver biopsy and in 170 relatives and neighbours. Levels of NTBI were correlated with indirect measures of iron status and conventional liver function tests. RESULTS: Non-transferrin-bound iron (> 2 micromol/L) was present in 43 people, 22 of patients of whom underwent liver biopsy and 21 relatives and neighbours. All but four of these had evidence of iron overload on the basis of either liver biopsy or elevated transferrin and serum ferritin concentrations. Among all 195 subjects, the presence of NTBI in serum was independently related to elevations in alanine and aspartate aminotransferase activity and bilirubin concentration. This relationship between serum NTBI and hepatic dysfunction was confirmed in the subgroup of 25 subjects with iron overload documented by liver biopsy. Non-transferrin-bound iron correlated significantly with elevations in alanine and aspartate aminotransferase activities after adjustment for hepatic iron grades, inflammation and diet. CONCLUSIONS: Non-transferrin-bound iron was found to be commonly present in African patients with dietary iron overload and to correlate with transferrin saturation and serum ferritin concentration. The independent relationship between NTBI and elevated liver function tests suggests that it may be part of a pathway leading to hepatic injury.     

Non-transferrin bound iron in Thalassemia: differential detection of redox active forms in children and older patients

Non-transferrin bound iron (NTBI) is commonly detected in patients with systemic iron overload whose serum iron-binding capacity has been surpassed. It has been perceived as an indicator of iron overload, impending organ damage and a chelation target in poly-transfused thalassemia patients. However, NTBI is a heterogeneous entity comprising various iron complexes, including a significant redox-active and readily chelatable fraction, which we have designated as "labile plasma iron" (LPI). We found that LPI levels can be affected by plasma components such as citrate, uric acid, and albumin. However, the inclusion of a mild metal mobilizing agent in the LPI assay (designated here as "eLPI"), at concentrations that do not affect transferrin-bound iron, largely overcomes such effects and provides a measure of the full NTBI content. We analyzed three distinct groups of poly-transfused, iron overloaded thalassemia patients: non-chelated children (3-13 yrs, Gaza, Palestine), chelated adolescents-young adults (13-28 yrs, Israel), and chelated adults (27-61 yrs, Israel) for LPI and eLPI. The eLPI levels in all three groups were roughly commensurate (r(2) = 0.61-0.75) with deferrioxamine-detectable NTBI, i.e., DCI. In older chelated patients, eLPI levels approximated those of LPI, but in poly-transfused unchelated children eLPI was notably higher than LPI, a difference attributed to plasma properties affected by labile iron due to lack of chelation, possibly reflecting age-dependent attrition of plasma components. We propose that the two formats of NTBI measurement presented here are complementary and used together could provide more comprehensive information on the forms of NTBI in patients and their response to chelation.     

Levels of non-transferrin-bound iron as an index of iron overload in patients with thalassaemia intermedia

Non-transferrin-bound iron (NTBI) was evaluated as an index of iron overload in a cross-sectional randomised study in 74 non-transfused patients with thalassaemia intermedia (TI). Mean NTBI (2·92 ± 3·43 μmol/l), serum ferritin (1023 ± 780 ng/ml) and liver iron concentration (LIC; 9·0 ± 7·4 mg Fe/g dry weight) were increased above reference-range levels. Significant positive correlations occurred between mean NTBI and LIC (Pearson correlation 0·36; P  = 0·002) and serum ferritin (Pearson correlation 0·421; P  < 0·0001); with higher levels observed in splenectomised patients. NTBI assessment has potential as a simple reliable approach to determining iron status in TI.     

Non‐transferrin bound labile plasma iron and iron overload in Sickle Cell Disease: a comparative study between Sickle Cell Disease and β thalassemic patients

Background: Blood transfusions are the standard of care in β thalassemia and transfusions are also indicated in sickle cell disease (SCD) patients with hypersplenism, recurrent vaso‐occlusive crises and for stroke prevention. Iron overload caused by blood transfusions in thalassemia and in SCD may affect morbidity and mortality. Recent studies of iron overload in SCD suggest that the biologic features of SCD and the chronic inflammatory state may protect SCD patients from iron damage. Designs and methods: In view of the controversy regarding the effect of iron overload in patients with SCD we studied the iron status, including non‐transferrin bound iron (NTBI) and labile plasma iron (LPI) levels in a cohort of 36 SCD patients and compare the results with 43 thalassemia patients. Results: Our results indicate that none of the SCD patients had clinical symptoms of iron overload. Only two SCD patients had NTBI values in the gray zone (0.4 units) and none had positive values. By contrast, 14 patients with thalassemia major and three with thalassemia intermedia had NTBI values above 0.6, level that are in the positive pathological range. Similarly, four thalassemia patients, but only one SCD patient had positive LPI levels. Conclusions: The parameters of iron status in SCD patients, even after frequent transfusions are different when compared to patients with thalassemia. The low NTBI and LPI levels found in patients with SCD are in keeping with the absence of clinical signs of iron overload in this disease.     

Vitamin E correlates inversely with non-transferrin-bound iron in sickle cell disease

Decreased serum vitamin E levels are found in homozygous sickle cell disease (SCD). Excessive transfusions may lead high non-transferrin-bound iron (NTBI). Hypothesizing a relationship between the two, vitamin E (measured using high performance liquid chromatography) was significantly lower in 30 SCD patients than in 30 age-/sex-matched controls (P < 0.001), but NTBI (bleomycin assay) was higher (P < 0.001). Vitamin E was lower in 10 transfused patients than in 20 non-transfused patients (P < 0.001) with a significant inverse correlation between the NTBI and vitamin E (r = -0.58, P < 0.001). NTBI associated with iron overload in SCD may increase the potential for oxidative damage and low vitamin E activity may compound this effect.     

The assessment of serum nontransferrin-bound iron in chelation therapy and iron supplementation

Nontransferrin-bound iron (NTBI) appears in the serum of individuals with iron overload and in a variety of other pathologic conditions. Because NTBI constitutes a labile form of iron, it might underlie some of the biologic damage associated with iron overload. We have developed a simple method for NTBI determination, which operates in a 96-well enzyme-linked immunosorbent assay format with sensitivity comparable to that of previous assays. A weak ligand, oxalic acid, mobilizes the NTBI and mediates its transfer to the iron chelator deferoxamine (DFO) immobilized on the plate. The amount of DFO-bound iron, originating from NTBI, is quantitatively revealed in a fluorescence plate reader by the fluorescent metallosensor calcein. No NTBI is found in normal sera because transferrin-bound iron is not detected in the assay. Thalassemic sera contained NTBI in 80% of the cases (range, 0.9-12.8 micromol/L). In patients given intravenous infusions of DFO, NTBI initially became undetectable due to the presence of DFO in the sera, but reappeared in 55% of the cases within an hour of cessation of the DFO infusion. This apparent rebound was attributable to the loss of DFO from the circulation and the possibility that a major portion of NTBI was not mobilized by DFO. NTBI was also found in patients with end-stage renal disease who were treated for anemia with intravenous iron supplements and in patients with hereditary hemochromatosis, at respective frequencies of 22% and 69%. The availability of a simple assay for monitoring NTBI could provide a useful index of iron status during chelation and supplementation treatments. (Blood. 2000;95:2975-2982)     

Iron overload across the spectrum of non‐transfusion‐dependent thalassaemias: role of erythropoiesis,splenectomy and transfusions

Non‐transfusion‐dependent thalassaemias (NTDT ) encompass a spectrum of anaemias rarely requiring blood transfusions. Increased iron absorption, driven by hepcidin suppression secondary to erythron expansion, initially causes intrahepatic iron overload. We examined iron metabolism biomarkers in 166 NTDT patients with β thalassaemia intermedia (n  = 95), haemoglobin (Hb) E/β thalassaemia (n  = 49) and Hb H syndromes (n  = 22). Liver iron concentration (LIC ), serum ferritin (SF ), transferrin saturation (TfSat) and non‐transferrin‐bound iron (NTBI ) were elevated and correlated across diagnostic subgroups. NTBI correlated with soluble transferrin receptor (sTfR ), labile plasma iron (LPI ) and nucleated red blood cells (NRBC s), with elevations generally confined to previously transfused patients. Splenectomised patients had higher NTBI , TfSat, NRBC s and SF relative to LIC , than non‐splenectomised patients. LPI elevations were confined to patients with saturated transferrin. Erythron expansion biomarkers (sTfR , growth differentiation factor‐15, NRBC s) correlated with each other and with iron overload biomarkers, particularly in Hb H patients. Plasma hepcidin was similar across subgroups, increased with >20 prior transfusions, and correlated inversely with TfSat, NTBI , LPI and NRBC s. Hepcidin/SF ratios were low, consistent with hepcidin suppression relative to iron overload. Increased NTBI and, by implication, risk of extra‐hepatic iron distribution are more likely in previously transfused, splenectomised and iron‐overloaded NTDT patients with TfSat >70%.     

Increased levels of advanced glycation end products positively correlate with iron overload and oxidative stress markers in patients with β-thalassemia major

The impaired biosynthesis of the β-globin chain in β-thalassemia leads to the accumulation of unpaired alpha globin chains, failure in hemoglobin formation, and iron overload due to frequent blood transfusion. Iron excess causes oxidative stress and massive tissue injuries. Advanced glycation end products (AGEs) are harmful agents, and their production accelerates in oxidative conditions. This study was conducted on 45 patients with major β-thalassemia who received frequent blood transfusions and chelation therapy and were compared to 40 healthy subjects. Metabolic parameters including glycemic and iron indices, hepatic and renal functions tests, oxidative stress markers, and AGEs (carboxymethyl-lysine and pentosidine) levels were measured. All parameters were significantly increased in β-thalassemia compared to the control except for glutathione levels. Blood glucose, iron, serum ferritin, non-transferrin-bound iron (NTBI), MDA, soluble form of low-density lipoprotein receptor, glutathione peroxidase, total reactive oxygen species (ROS), and AGE levels were significantly higher in the β-thalassemia patients. Iron and ferritin showed a significant positive correlation with pentosidine (P?<?0.01) but not with carboxymethyl-lysine. The NTBI was markedly increased in the β-thalassemia patients, and its levels correlated significantly with both carboxymethyl-lysine and pentosidine (P?<?0.05). Our findings confirm the oxidative status generated by the iron overload in β-thalassemia major patients and highlight the enhanced formation of AGEs, which may play an important role in the pathogenesis of β-thalassemia major.     

Labile plasma iron in iron overload: redox activity and susceptibility to chelation

Plasma non-transferrin-bound-iron (NTBI) is believed to be responsible for catalyzing the formation of reactive radicals in the circulation of iron overloaded subjects, resulting in accumulation of oxidation products. We assessed the redox active component of NTBI in the plasma of healthy and beta-thalassemic patients. The labile plasma iron (LPI) was determined with the fluorogenic dihydrorhodamine 123 by monitoring the generation of reactive radicals prompted by ascorbate but blocked by iron chelators. The assay was LPI specific since it was generated by physiologic concentrations of ascorbate, involved no sample manipulation, and was blocked by iron chelators that bind iron selectively. LPI, essentially absent from sera of healthy individuals, was present in those of beta-thalassemia patients at levels (1-16 microM) that correlated significantly with those of NTBI measured as mobilizer-dependent chelatable iron or desferrioxamine chelatable iron. Oral treatment of patients with deferiprone (L1) raised plasma NTBI due to iron mobilization but did not lead to LPI appearance, indicating that L1-chelated iron in plasma was not redox active. Moreover, oral L1 treatment eliminated LPI in patients. The approach enabled the assessment of LPI susceptibility to in vivo or in vitro chelation and the potential of LPI to cause tissue damage, as found in iron overload conditions.     

Differential accumulation of non-transferrin-bound iron by cardiac myocytes and fibroblasts

Cardiac myocytes accumulate iron preferentially over fibroblast-like non-myocytes, both in clinical iron overload and when the cells are grown together in culture. In order to determine whether this reflects the tissue context or is an inherent property of the cells, we studied iron transporters, transport kinetics, and iron efflux in homogeneous cultures of rat cardiac myocytes and fibroblasts. In both cells, the rate of uptake of 59Fe from transferrin was insignificant, compared to the rate of uptake from non-transferrin-bound iron (NTBI). Expression of transferrin receptor mRNA and protein, and divalent metal transporter 1 (DMT1) mRNA, could not account for any difference in iron accumulation, and proportional efflux after iron loading was similar in both cells. Nevertheless, iron accumulation from NTBI over 72 h was greater in myocytes as determined by histological staining and quantitative iron measurement. NTBI uptake was greater for Fe2+ than Fe3+ in both cells, was increased by iron loading in both cells to a similar extent, and was characterized bysimilar Michaelis constants (K(m)) in all cases (redox state and presence or absence of iron loading). However, V(max) values were about 10-fold higher in myocytes. We conclude that preferential iron accumulation in cardiac myocytes, compared to fibroblasts, is due to a higher capacity of the NTBI-transporter system, and reflects an inherent difference in NTBI acquisition by the individual cell types.     

Mechanisms of plasma non‐transferrin bound iron generation: insights from comparing transfused diamond blackfan anaemia with sickle cell and thalassaemia patients

In transfusional iron overload, extra‐hepatic iron distribution differs, depending on the underlying condition. Relative mechanisms of plasma non‐transferrin bound iron (NTBI) generation may account for these differences. Markers of iron metabolism (plasma NTBI, labile iron, hepcidin, transferrin, monocyte SLC40A1 [ferroportin]), erythropoiesis (growth differentiation factor 15, soluble transferrin receptor) and tissue hypoxia (erythropoietin) were compared in patients with Thalassaemia Major (TM), Sickle Cell Disease and Diamond‐Blackfan Anaemia (DBA), with matched transfusion histories. The most striking differences between these conditions were relationships of NTBI to erythropoietic markers, leading us to propose three mechanisms of NTBI generation: iron overload (all), ineffective erythropoiesis (predominantly TM) and low transferrin‐iron utilization (DBA).     

High nontransferrin bound iron levels and heart disease in thalassemia major

Although the presence of nontransferrin bound plasma iron (NTBI) in transfusional iron overload is well documented, knowledge about its clinical significance is limited. We assessed NTBI levels in a large and homogeneous series of thalassemia patients on regular transfusion and chelation and explored the hypothesis that NTBI levels may be associated with relevant clinical outcomes: in particular, heart disease. Among 174 patients with thalassemia major and intermedia, we showed the presence NTBI in 145 of 174 or 83.3% of cases. NTBI levels correlated with transferrin saturation, age, and ALT, and not with serum ferritin or liver iron concentrations. At a multiple regression analysis, transferrin saturation and heart disease but not age was independent predictors of NTBI. Patients with heart disease had NTBI levels significantly higher than those without. All patients with heart disease had transferrin saturation above 70%, and all were NTBI positive. Conversely, none of the patients without NTBI and/or with transferrin saturation less than 70% had preclinical or clinical heart disease. To our knowledge, this is the first documentation of a link between the presence of NTBI in thalassemic patients with transfusional iron overload and heart disease. Further investigation from these preliminary findings may clarify whether NTBI assessment may have a role in evaluating the risks and optimizing treatment for transfusion‐dependent patients. Am. J. Hematol., 2009. © 2008 Wiley‐Liss, Inc.     

Absence of cardiac siderosis by MRI T2* despite transfusion burden,hepatic and serum iron overload in Lebanese patients with sickle cell disease

Background: The use of magnetic resonance imaging (MRI) to detect organ‐specific iron overload is becoming increasingly common. Although hepatic iron overload has been recognized in patients with sickle cell disease (SCD), cardiac iron deposition has only been examined in a few reports. Methods: This was a cross‐sectional study of 23 patients with SCD. Patient charts were reviewed and data collected for drug use, total lifetime transfusions (TLT), transfusion rate (TR), status of the spleen, and comorbid illnesses or infections. Blood samples were obtained for assessment of hemoglobin, serum ferritin, non‐transferrin‐bound iron (NTBI), and liver enzyme levels. Doppler echocardiography was performed to detect pulmonary hypertension (PHT) and assess left ventricular ejection fraction. Cardiac iron levels were measured by MRI T2*. Direct determination of liver iron concentration (LIC) was performed using R2 MRI. In this study, cardiac T2* >20 ms was considered normal. Results: The mean age was 24.4 ± 9.7 yr, with a male to female ratio of 15:8. A total of 9 (49.9%) patients were splenectomized. The mean TR was 14.1 ± 13.2 Units/yr, and the mean hemoglobin level was 9.0 g/dL. PHT was detected in 6 (27.3%) patients, but none had evidence of heart failure. The mean serum ferritin, LIC, and NTBI levels were 997.7 ng/mL, 4.6 mg Fe/g dw, and 1.1 ± 2.2, respectively. TR was a much better predictor of iron burden (LIC, ferritin, NTBI) than TLT. In fact, TR less than 10 Units/yr did not produce significant iron overload reflecting spontaneous losses as high as 0.11 mg/kg/d. None of the patients had evidence of cardiac iron overload (mean cardiac T2* = 37.3 ± 6.2 ms; range: 21.9–46.8 ms). There was also no statistically significant correlation between cardiac T2* values and any of the study variables. Conclusion: Our study demonstrates that TR is a stronger predictor of iron overload than TLT. It also confirms cardiac sparing in patients with SCD, even in subjects with significant transfusion burden, systemic and hepatic iron overload.     

Pro-oxidative/antioxidative imbalance: a key indicator of adverse outcome in hematopoietic stem cell transplantation

Introduction: Pretransplantation iron overload (IO) is considered as a predictor of adverse outcome in hematopoietic stem cell transplantation (HSCT). Peroxidative tissue injury caused by IO leads to progressive organ dysfunction. Methods: This is a retro‐prospective study which explores the possible relationship between IO, oxidative stress and transplant outcome. Serum samples of 149 consecutive HSCT candidates were subjected to analysis of iron parameters, including nontransferrin bound iron (NTBI) and pro‐oxidant/antioxidant status. Results: Serum ferritin was found to be positively correlated with NTBI and negatively correlated with glutathione peroxidase (GPx) and superoxide dismutase (SOD). An inverse correlation of NTBI with SOD, total antioxidant potential (TAP) and malonyldialdehide (MDA) was also demonstrated. An adverse impact of serum ferritin level on early posttransplant complications including pulmonary toxicity, fungal infections and sinusoidal obstruction syndrome (SOS) was shown. A significant impact of NTBI on +30 day (P = 0.027) and +100 day survival (P = 0.028) was shown in auto‐transplanted patients. MDA levels had a significant impact on +30 day and +100 day survival in autologous (P = 0.047; P = 0.026) and allogeneic (P = 0.053; P = 0.059) groups. GPx (P = 0.016) and MDA (P = 0.021) were identified as independent prognostic parameters for overall survival in allo‐transplanted patients. Conclusion: Pretransplantation IO might be a major contributor to adverse outcome in HSCT recipients through an impaired pro‐oxidative/antioxidative homeostasis. The reversible nature of IO and oxidative stress suggests that early preventive strategies might have a potential to improve transplant outcome.     

Calcium channels and iron uptake into the heart

Iron overload can lead to iron deposits in many tissues,particularly in the heart.It has also been shown to be associated with elevated oxidative stress in tissues.Elevated cardiac iron deposits can lead to iron overload cardiomyopathy,a condition which provokes mortality due to heart failure in iron-overloaded patients.Currently,the mechanism of iron uptake into cardiomyocytes is still not clearly understood.Growing evidence suggests L-type Ca2+channels(LTCCs)as a possible pathway for ferrous iron(Fe2+)uptake into cardiomyocytes under iron overload conditions.Nevertheless,controversy still exists since some findings on pharmacological interventions and those using different cell types do not support LTCC’s role as a portal for iron uptake in cardiac cells.Recently,T-type Ca2+channels (TTCC)have been shown to play an important role in the diseased heart.Although TTCC and iron uptake in cardiomyocytes has not been investigated greatly,a recent finding indicated that TTCC could be an important portal in thalassemic hearts.In this review,comprehensive findings collected from previous studies as well as a discussion of the controversy regarding iron uptake mechanisms into cardiomyocytes via calcium channels are presented with the hope that understanding the cellular iron uptake mechanism in cardiomyocytes will lead to improved treatment and prevention strategies,particularly in iron-overloaded patients.     

Serum non-transferrin-bound iron in beta-thalassaemia major patients treated with desferrioxamine and L1

Non-transferrin-bound iron (NTBI) in plasma is toxic due to its ability to participate in free radical formation with resultant peroxidation and damage to cell membranes and other biomolecules. NTBI concentration was determined in serum in 12 normal volunteers and in 52 patients with beta-thalassaemia major by a modification of the method described by Singh et al (1990). There was no detectable NTBI in normal individuals. In the patients NTBI values ranged from -1.5 to 9.0 mumol/l (mean +/- SD: 3.6 +/- 2.3). The patients' serum ferritin concentrations ranged from 207 to 11,400 micrograms/l (2674 +/- 2538), total serum iron from 20 to 61 mumol/l (39.5 +/- 9.6) and transferrin saturation from 44 to 110% (84.5 +/- 13.8). The NTBI correlated significantly with serum ferritin (r = 0.467, P < 0.001), total serum iron (r = 0.608, P < 0.001) and transferrin saturation (r = 0.481, P < 0.005). When patients were grouped according to their compliance with desferrioxamine (DFX) therapy, the good compliers had significantly lower NTBI concentrations compared to the poor compliers (poor: 5.4 +/- 1.8 mumol/l v good: 2.7 +/- 1.7 mumol/l, P < 0.001). There was also a significant difference between the level of NTBI and whether or not the patients had complications of iron overload (5.2 +/- 1.7 mumol/l v 2.9 +/- 1.6 mumol/l, P < 0.001). During this study 10 patients were entered into a trial of the oral iron chelator 1,2- dimethyl-3-hydroxypyrid-4-one (L1). Their NTBI values were observed during the first 6 months of the trial and showed a significant fall (paired t-test: P = 0.007). These results suggest that the level of NTBI may prove helpful in assessing the efficiency of chelation in patients with transfusion dependent anaemia and help to predict organ damage.     

Determinants of iron status and bilirubin levels in congenital dyserythropoietic anaemia type I

Seven untransfused patients with congenital dyserythropoietic anaemia type I were investigated to assess the determinants of both iron overload and serum bilirubin levels. The serum ferritin concentration was increased in all patients and non-transferrin-bound iron (NTBI) was increased in all but one patient. None of the patients showed the C282Y mutation in the hereditary haemochromatosis gene, HFE. One patient was homozygous for the H63D mutation in this gene. The data indicated that differences in the extent of iron overload were not mediated by co-inheritance of the C282Y mutation in the HFE gene but could largely be explained by differences in the severity of anaemia and ineffective erythropoiesis, and in the age of the patient. In one patient an unusually high plasma bilirubin level was associated with the variant A[TA]7TAA configuration in the TATA box of the uridine diphosphate glucuronosyltransferase (UGT-1A) gene promoter, the mutation found in most patients with mild Gilbert's syndrome.