Desferrioxamine-chelatable Iron Research Articles

The ability to determine the non-transferrin-bound iron and total iron in the human placenta using high-performance liquid chromatography method

Iron is one of the most important microelements in the human body. It is a component of haemoglobin, which transports oxygen to all cells in the organism. It is also used in the synthesis of myelin, neurotrans-mitters, and DNA and transfers electrons in biochemical reactions. Iron is also responsible for regular development of the foetus’ central nervous system. Furthermore, as a result of Fenton reactions, iron leads to formation of toxic free radicals. The existence of non-transferrin-bound iron (NTBI) and its part desfer-rioxamine-chelatable iron (DCI) can be used to assess this element in the body. The placenta is an organ transition that is formed during pregnancy in the female organism. It has a dense web of blood vessels in which dynamic exchange of blood between mother and foetus takes place. As a result, a fraction of NTBI may be present in the placenta. The main goal of this work was to develop a method for determining total iron and desferrioxamine-chelatable iron in solid tissues - the human placenta.

Ability to determine the desferrioxamine‐chelatable iron fractions of nontransferrin‐bound iron using HPLC

Iron is an essential element in human development. It is imperative for oxygen and electron transport and also for DNA and neurotransmitters synthesis. On the other hand, this metal is able to participate in Fenton's reaction that in turn leads to free radical damage. The most toxic fraction of iron - nontransferrin-bound iron and its part desferrioxamine-chelatable iron - can serve as an exquisite biomarker in the identification of iron imbalance. The goal of the present study was to devise a simple, repeatable, and inexpensive method for the determination of desferrioxamine-chelatable iron in serum blood samples. The assay procedure is based on desferrioxamine complex formation with iron ions followed to ferrioxamine and its quantitative measurement using RP-HPLC method. The desferrioxamine-chelatable iron was extracted from blood by centrifugation and SPE method. Chromatographic separation was performed at 40°C by step-form gradient elution using Cadenza CD-C18 column (150 × 4.6 mm id, particle size of 3.0 μm) connected with precolumn for contaminants removal. Gradient HPLC elution has been carried out with solvent A (10 mM Tris-HCl, pH 5.5) and solvent B (ACN). The flow rate was 1.2 mL/min, and the total separation time was 5 min. The linear quantitation range was 2.5-500 μM (r = 0.9973), and the LOD and LOQ were 0.42 and 1.29 μM, respectively. Proposed HPLC method allowed for the determination of desferrioxamine-chelatable iron fraction's of nontransferrin-bound iron, both in the buffer and the serum supplemented with iron ions as well as in the patients' serum samples with good results of precision and recovery. The developed method found to be sufficiently precise and reproducible for established conditions and after validation and may be used for routine assay of desferrioxamine-chelatable iron in biological samples.

Hypoxia-Induced Post-Translational Changes in Red Blood Cell Protein Map of Newborns

Tyrosine (Tyr) phosphorylation is implicated in the modification of several erythrocyte functions, such as metabolic pathways and membrane transport, as well as in signal transduction systems. Here we describe the map of Tyr-phosphorylated soluble proteins of newborn red blood cells (RBC) using an in vitro model simulating RBC reoxygenation at birth after an intrauterine hypoxic event. We tested the hypothesis that a hypoxic environment and subsequent reoxygenation promote post-translational changes in the RBC protein map of newborns, in addition to desferrioxamine (DFO)-chelatable iron (DCI) release and methemoglobin (MetHb) formation. Umbilical cord blood RBC were incubated under hypoxic conditions for 16 h at 37 degrees C, and subsequently for 8 h under aerobic conditions. Control erythrocytes were incubated under aerobic conditions at 37 degrees C for the period of the experiment, i.e. for 24 h. Tyr-phosphorylation proteins were assessed using advanced high-resolution two-dimensional electrophoresis, 2-D immunoblot analysis with anti-phosphotyrosine (anti-pTyr) antibodies, and computer-aided electrophoretogram analysis. Higher DCI release and MetHb formation were observed in newborn RBC incubated under hypoxic conditions than in those incubated aerobically. Different immunoreactivity patterns with anti-pTyr antibodies were also observed between newborn RBC incubated under hypoxic conditions and controls. A hypoxic environment is a factor promoting DCI release, a well-known condition of oxidative stress. This is the first map of Tyr-phosphorylated soluble proteins of newborn RBC obtained using an in vitro model simulating RBC reoxygenation at birth after an intrauterine hypoxic event. Our results suggest that hypoxia increases Tyr-phosphorylation of antioxidant proteins, protecting RBC against oxidative stress.

Plasma F 2-isoprostanes are elevated in newborns and inversely correlated to gestational age

F 2-isoprostanes, prostaglandin F 2-like compounds formed by free radical-catalyzed lipid peroxidation, are considered the most reliable markers of oxidative stress. It has been repeatedly suggested that newborns are exposed to conditions of oxidative stress resulting from the change from a low oxygen pressure in utero to a high oxygen pressure at birth. We measured the levels of F 2-isoprostanes in plasma of newborns by gas chromatography/mass spectrometry and we found that F 2-isoprostanes are significantly higher in term newborns compared to healthy adults. The greatest values were found in preterm newborns in whom F 2-isoprostanes are even higher than in term babies. Moreover a significant inverse correlation was found between the plasma levels of isoprostanes and the gestational age. A quite normal level of isoprostanes was found in the mothers both at delivery and during pregnancy. Placental total F 2-isoprostanes (sum of free plus esterified) were significantly higher in preterm compared to term deliveries and such a difference might account for the difference in plasma isoprostanes. Plasma non-protein-bound iron is higher in preterm than in term newborns, even if no correlation was found with plasma F 2-isoprostanes. Erythrocyte desferrioxamine-chelatable iron content (0 time) and release (24 h of aerobic incubation) are higher in newborns than in adults and in preterm than in term newborns, but again no correlation was found with plasma F 2-isoprostanes. The marked increase in plasma isoprostanes suggests that oxidative stress is a feature of the physiopathological changes seen in the perinatal period.

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

AbstractPlasma 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 β-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 β-thalassemia patients at levels (1-16 μM) 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. (Blood. 2003;102:2670-2677)

Iron-induced oxidant stress in nonparenchymal liver cells: mitochondrial derangement and fibrosis in acutely iron-dosed gerbils and its prevention by silybin.

Hepatic fibrosis due to iron overload is mediated by oxidant stress. The basic mechanisms underlying this process in vivo are still little understood. Acutely iron-dosed gerbils were assayed for lobular accumulation of hepatic lipid peroxidation by-products, oxidant-stress gene response, mitochondrial energy-dependent functions, and fibrogenesis. Iron overload in nonparenchymal cells caused an activation of hepatic stellate cells and fibrogenesis. Oxidant-stress gene response and accumulation of malondialdehyde-protein adducts were restricted to iron-filled nonparenchymal cells, sparing nearby hepatocytes. Concomitantly, a significant rise in the mitochondrial desferrioxamine-chelatable iron pool associated with the impairment of mitochondrial oxidative metabolism and the hepatic ATP decrease, was detected. Ultrastructural mitochondrial alterations were observed only in nonparenchymal cells. All biochemical and functional derangements were hindered by in vivo silybin administration which blocked completely fibrogenesis. Iron-induced oxidant stress in nonparenchymal cells appeared to bring about irreversible mitochondrial derangement associated with the onset of hepatic fibrosis.

Desferrioxamine-chelatable iron, a component of serum non–transferrin-bound iron, used for assessing chelation therapy: Presented in preliminary form at the 10th International Conference on Oral Chelators symposium on iron chelators held in Limassol, Cyprus, March 2000 (Transfusion Science, in press).

AbstractThis study introduces a method for monitoring a component of serum non–transferrin-bound iron (NTBI), termed “desferrioxamine-chelatable iron” (DCI). It is measured with the probe fluorescein-desferrioxamine (Fl-DFO), whose fluorescence is stoichiometrically quenched by iron. DCI was found in the serum of most patients with thalassemia major (21 of 27 tested, range 1.5-8.6 μM), but only in a minority of patients with hereditary hemochromatosis (8 of 95 samples from 39 patients, range 0.4- 1.1 μM) and in none of 48 controls. The method was applied to monitoring the appearance of iron in the serum of patients under chelation therapy. Short-term (2 hours) follow-up of patients immediately after oral administration of deferriprone (L1) showed substantial mobilization of DCI into the serum (up to10 μM within 30-60 minutes). The transfer of DCI from L1 to Fl-DFO was observed in vitro with preformed L1-iron complexes, and occurred even at L1/iron ratios exceeding 3:1. Simultaneous administration of oral L1 and intravenous DFO to patients abrogated the L1-mediated rise in DCI, consistent with the shuttling of iron from L1 to DFO in vivo. A similar iron transfer from L1 to apo-transferrin was observed in vitro, lending experimental support to the notion that L1 can shuttle iron in vivo to other high-affinity ligands. These results provide a rationale for using chelator combinations, with the highly permeant L1 acting as an intracellular chelator-shuttle and the less permeant DFO serving as an extracellular iron sink. Potential applications of the DCI assay may be for studying chelator action and as an index of patient chelation status.

Desferrioxamine-chelatable iron (DCI), a component of serum non-transferrin-bound iron (NTBI) used for assessing iron chelation therapy

Desferrioxamine-chelatable iron (DCI), a component of serum non-transferrin-bound iron (NTBI) used for assessing iron chelation therapy

Iron-induced oxidant stress leads to irreversible mitochondrial dysfunctions and fibrosis in the liver of chronic iron-dosed gerbils. The effect of silybin.

Hepatic iron toxicity because of iron overload seems to be mediated by lipid peroxidation of biological membranes and the associated organelle dysfunctions. However, the basic mechanisms underlying this process in vivo are still little understood. Gerbils were dosed with weekly injections of iron-dextran alone or in combination with sylibin, a well-known antioxidant, by gavage for 8 weeks. A strict correlation was found between lipid peroxidation and the level of desferrioxamine chelatable iron pool. A consequent derangement in the mitochondrial energy-transducing capability, resulting from a reduction in the respiratory chain enzyme activities, occurred. These irreversible oxidative anomalies brought about a dramatic drop in tissue ATP level. The mitochondrial oxidative derangement was associated with the development of fibrosis in the hepatic tissue. Silybin administration significantly reduced both functional anomalies and the fibrotic process by chelating desferrioxamine chelatable iron.

The role of desferrioxamine chelatable iron in rat liver mitochondrial dysfunction in chronic dietary iron overload

Lipid peroxidation and organelle dysfunction are important factors in hepatic iron toxicity. The form of the intracellular iron responsible for these abnormalities is still unknown. In order to investigate the iron species inducing cell injury, the level of chelatable iron in the liver mitochondria isolated from rats fed a 2.5% carbonyl iron diet for 12 weeks was measured by EPR spectroscopy. The presence of lipid peroxidation products and the energy transducing capacility of the mitochondrial inner membrane was evaluated in parallel. The total iron concentration in the liver mitochondria from iron fed rats progressively increased up to 6 weeks, almost reaching a steady-state. By contrast the level of chelatable iron in mitochondrial fraction transiently increased at about 3–6 weeks of treatment. The induction of lipid peroxidation and a large decrease of ATP occurred at the same time. The enhancement of the energy dissipating calcium cycling was in parallel revealed by studying the mitochondria membrane potential. These results gave experimental evidence to the proposal that the chelatable iron level plays a critical role in initiating organelle dysfunction, at least in this experimental model.

Role of iron in ischemia-reperfusion oxidative injury of rat lungs.

We have previously shown that isolated, oxygen-ventilated rat lungs generate reactive oxygen species during ischemia and reperfusion. To evaluate the role of free iron in lung ischemia/reperfusion injury, we measured desferrioxamine-chelatable iron (DC-Fe), protein carbonyls, and thiobarbituric acid reactive substances (TBARS) in lungs subjected to global ischemia and reperfusion. There were no changes in DC-Fe, TBARS, or protein carbonyls during 2 h of control perfusion. There was a 2.3-fold increase in DC-Fe during 1 h ischemia and a further doubling during reperfusion. Results were similar when lungs were ventilated with O2 or N2. Lung TBARS increased 214% during ischemia/reperfusion with oxygen ventilation; unlike DC-Fe release, this change was blocked by N2 ventilation. Protein carbonyl content of the lung also increased significantly (113%) with ischemia/reperfusion. Ferric 8-hydroxyquinoline added to the perfusate before ischemia increased DC-Fe in lung tissue and significantly enhanced the lipid and protein oxidation of ischemic/reperfused lungs. The added perfusate iron had no effect on control lungs. Proteins isolated from the iron-supplemented ischemic/reperfused lungs were resistant to further in vitro oxidation induced by hydrogen peroxide (30 microM) whereas proteins from control perfused lungs were oxidized under these conditions. These results indicate that DC-Fe plays an important role in lung oxidative injury with ischemia/reperfusion. DC-Fe is released from iron-storage sites during ischemia and promotes tissue oxidation when oxygenation is maintained during ischemia or restored during reperfusion.

Serum indicators of free radical activity in idiopathic pulmonary fibrosis.

Serum levels of free radical activity were measured in 37 patients with idiopathic pulmonary fibrosis (IPF) and 16 control subjects. Three assays used were (1) simultaneously measured levels of the 9,11-diene conjugate of linoleic acid and 9,12-linoleic acid expressed as a percent molar ratio (%MR), a measure of free-radical-mediated lipid peroxidation; (2) thiobarbituric acid reactive substances (TBARS), one of which is malondialdehyde; (3) desferrioxamine-chelatable iron assay, a measure of the potential iron available to catalyze free radical generation. Mean %MR, TBARS and desferrioxamine-chelatable iron were all elevated initially in patients with IPF compared with control subjects (%MR, p < 0.0001; TBARS, p = 0.0013; desferrioxamine-chelatable iron, p = 0.0029). Furthermore, the serum %MR was higher in a subset of patients with clinically worsening IPF than in those patients with clinically stable disease (p = 0.002). Treatment did not appear to affect the three different serum indicators of free radical activity. Thus, lipid peroxidation appears to be increased in patients with IPF and is associated with an increase in desferrioxamine-chelatable iron levels. Serum % MR levels appeared to correlate with clinical disease activity, and they may have a role in monitoring disease activity.

Indicators of free radical activity in patients developing radiation pneumonitis

Purpose : Radiation pneumonitis is thought to occur as the result of excess free radical generation following radiotherapy. Various in vitro studies have shown that large doses of irradiation can cause membrane lipid peroxidation and the oxidation of protein sulphuryl groups. We, therefore, studied two circulating markers of lipid peroxidation and an indicator of “catalytic iron” (potentially available iron to catlyze the generation of free radicals) in patients undergoing radiotherapy. Methods and Materials : The 9,11 diene conjugate of 9,12 linoleic acid, expressed as their molar ratio (percentage molar ratio (MR)) and thiobarbituric acid reactive acid-substances (TBARS), as well as levels of circulating desferrioxamine-chelatable iron assay, were assayed. Serial blood samples were taken over a 3-month period in 25 patients with inoperable nonsmall cell lung cancer. Results : Ten patients developed radiation pneumonitis. The patients who developed pneumonitis showed a tendency for the serum percentage molar ratio to increase after a week. The change in the percentage molar ratio between Time 0 and 1 week of radiotherapy was significantly higher in the group that subsequently developed pneumonitis compared to the group that did not ( p = 0.002). The initial serum TBARS levels in patients were not significantly elevated compared to controls and there was no difference in the serum TBARS levels in the pneumonitis and nonpneumonitis groups throughout the study period. After 1 week of radiotherapy the group that subsequently developed pneumonitis had a significantly higher level of desferrioxamine-chelatable iron (DFx-iron) compared with the nonpneumonitis group ( p = 0.05). Conclusion : These data suggest that both the percentage MR and DFx-iron appear to reflect an increased susceptibility to develop radiation pneumonitis and after 1 week of radiotherapy they indicate patients who are likely to subsequently develop pneumonitis. Hence, these indicators could indicate the group of patients that could benefit from intervention therapies with antioxidants.

Circulating markers of free radical activity in patients with pulmonary tuberculosis

Setting: Toxic free radicals have been implicated in the development of lung fibrosis which may be a long-term sequela of pulmonary tuberculosis. Objective: To measure circulating indicators of free radical activity in patients with pulmonary tuberculosis in order to determine whether patients with active pulmonary TB have elevated levels of circulating free radical activity, and whether these levels correlated with disease activity as determined by other blood markers of inflammation. Design: 17 patients with active pulmonary tuberculosis were studied. Serial serum levels of 3 assays of free radical activity were measured at diagnosis (17 patients), and over a 2–7 month period on chemotherapy (8 patients). 3 patients with active lymph node tuberculosis were also studied and 4 patients with old lung scarring from previously treated tuberculosis had their serum markers analysed. Results: All 3 serum markers of free radical activity were elevated in patients with active pulmonary tuberculosis. During serial measurement in 8 patients the % molar ratio of 9,11 linoleic acid/9,12 linoleic acid fell progressively with treatment. Thiobarbituric acid reactive substances (TBARS) were initially elevated in 6 8 patients and remained elevated despite treatment. In 2 patients TBARS were in the normal range at presentation but subsequently rose with treatment. Desferrioxamine-chelatable iron was initially normal in all but 1 patient, remained normal in 2 patients, rose in 4 patients and fell in 1 patient. Conclusions: These results suggest that increased circulating levels of free radical activity are found in active pulmonary tuberculosis and hence may play a role in the resultant fibrosis. It also reinforces the belief that a range of free radical activity (FRA) indicators are produced in any inflammatory process with fibrogenic potential and that these indicators may be measuring different stages of the disease process.

Iron absorption in normal subjects and patients with idiopathic haemochromatosis: relationship with serum ferritin concentration.

In 52 normal subjects there was an inverse relationship between serum ferritin concentration and iron absorption. In 21 measurements in 15 patients with idiopathic haemochromatosis there was a similar inverse relationship but absorption was higher in relation to iron stores at all levels. Haemochromatotic patients with normal serum ferritin levels had abnormally high values for desferrioxamine chelatable iron and there was no correlation between chelatable iron and iron absorption.

Studies on low dose chloroquine therapy and the action of chloroquine in symptomatic porphyria.

Summary.— Eight patients with symptomatic porphyria (porphyria cutanea tarda symptomatica, PCT) were treated with small doses of chloroquin sulphate over periods ranging from 2 to 16 weeks. A good clinical and biochemical response was obtained in all but one patient, who relapsed after initial improvement. Manifestations of acute untoward reaction were seen in one patient only. It is concluded that low dose chloroquine therapy is both safe and effective in PCT. Studies in porphyric rats indicate that the mechanism of action of chloroquine in PCT is probably not related, as has been suggested, to the formation of a chloroquine-porphyrin complex which damages liver cells and thereby leads to a reduction in hepatic porphyrin stores. Chloroquine therapy led to increased urinary iron excretion and an increase in the desferrioxamine-chelatable iron in patients with PCT. It is suggested that the prolonged beneficial effects of chloroquine treatment may be related to reduction in a specific pool of liver iron.

Desferrioxamine chelatable iron in haemolytic, megaloblastic and sideroblastic anaemias.

SUMMARYThe chelation of body iron by desferrioxamine methanesulphonate in patients with haemolytic anaemias of varying types, in megaloblastic and in sideroblastic anaemias was measured by the differential ferrioxamine test. Thirty‐five out of 44 patients studied had chelation values above the normal range, sometimes exceeding those found in untreated haemochromatosis.In the haemolytic anaemias, the amount of chelation increased with the severity of the anaemia. In the megaloblastic anaemias, specific therapy reduced the amount of iron chelated by desferrioxamine at the same time as normal erythropoiesis was re‐established.Concepts of labile iron and metabolically active iron are compared with desferrioxamine chelatable iron. These findings suggest that in addition to storage iron (ferritin‐haemosiderin) chelated with difficulty, there is a compartment of readily chelatable iron derived from haem catabolism, possibly sited in reticulo‐endothelium. A model of intracellular iron transport in reticulo‐endothelial cells is proposed which provides a primary pathway of metabolically active, highly chelatable iron derived from haemoglobin catabolism, and a secondary pathway to intracellular ferritin.