Complexation of iron(III) and iron(II) by citrate. Implications for iron speciation in blood plasma

Estimates of the concentrations and identity of the predominant complexes of iron with the low-molecular-mass ligands in vivo are important to improve current understanding of the metabolism of this trace element. These estimates require a knowledge of the stability of the iron-citrate complexes. Previous studies on the equilibrium properties of the Fe(III)-citrate and Fe(II)-citrate are in disagreement. Accordingly, in this work, glass electrode potentiometric titrations have been used to re-determine the formation constants of both the Fe(III)- and Fe(II)-citrate systems at 25 degrees C in 1.00 M (Na)Cl and the reliability of these constants has been evaluated by comparing the measured and predicted redox potentials of the ternary Fe(III)-Fe(II)-citrate system. The formation constants obtained in this way were used in computer simulation models of the low-molecular-mass iron fraction in blood plasma. Redox equilibria of iron are thus included in large models of blood plasma for the first time. The results of these calculations show the predominance of Fe(II)-carbonate complexes and a significant amount of aquated Fe(II) in human blood plasma.     

Identification and localization of low-molecular-mass GTP-binding proteins associated with synaptic vesicles and other membranes

GTP-binding proteins were studied in synaptic vesicles prepared from bovine brain by differential centrifugation and separated further from plasma membranes using gel permeation chromatography. Following separation by SDS-PAGE of proteins from the different fractions, and transfer to nitrocellulose sheets, the presence and localization of low-molecular-mass GTP-binding proteins were assessed by [alpha-32 P]GTP binding. The vesicle-membrane fraction (SV) was enriched in synaptophysin (p38, a synaptic vesicle marker) and contained low-molecular-mass GTP-binding proteins; these consisted of a major 27 kDa protein and minor components (Mr 26 and 24 kDa) which were trypsin-sensitive and immunologically distinguishable from ras p21 protein. GTP-binding proteins of low molecular mass, but displaying less sensitivity to trypsin, were also found in the plasma membrane fraction (PM; enriched in Na+/K(+)-ATPase). In addition, the PM fraction contained GTP-binding proteins with higher Mr (Gi alpha and G0 alpha), together with another GTP-binding protein, ras p21. Putative function(s) of these GTP-binding proteins with low mass are discussed.     

Antioxidant protection against organic and inorganic oxygen radicals by normal human plasma: the important primary role for iron-binding and iron-oxidising proteins.

Normal human plasma contains numerous high- and low-molecular-mass redox-active molecules that are able to react rapidly with organic and inorganic oxygen radicals. The ability of such plasma molecules to substantially inhibit, or delay, free-radical mediated oxidation of added substrates has led to their classification as important biological antioxidants. Using phospholipids to detect organic oxygen radicals and deoxyribose to detect inorganic oxygen radicals, we here show that the primary antioxidants of normal human plasma reside mainly in two plasma proteins representing no more than 4% of the total proteins present. The iron-binding properties of transferrin and the iron-oxidising properties of caeruloplasmin, at a reaction dilution of 1:50, offer considerable protection against organic and inorganic oxygen radicals generated by iron and ascorbate. Plasma thiol-group-containing molecules, at concentrations well below those that would be required to compete with the detector molecule (based on known second-order rate constants for reaction with hydroxyl radicals) inhibited damage to deoxyribose, but stimulated damage to phospholipids.     

Control of hemoglobin synthesis in erythroid differentiating K562 cells: II. Studies of iron mobilization in erythroid cells by high-performance liquid chromatography–electrochemical detection

We have demonstrated that iron controls hemoglobin (Hb) synthesis in erythroid differentiating K562 cells by enhancing the activity of a key enzyme of the Hb synthesis, δ-aminolevulinate synthase (ALAS). In the present study, we studied iron mobilization and the role of iron in erythroid differentiating cells by measuring the level of iron by means of high-performance liquid chromatography using electrochemical detection (HPLC–ED). After treatment of K562 cells with sodium butyrate, the expression of transferrin receptor (TfR) increased initially, followed by an increase in the levels of both total iron and Hb as well as the ALAS activity. However, no increase could be found in the levels of non-heme iron, low-molecular-mass iron (LMMFe) and ferritin. Addition of diferric transferrin (FeTf) enhanced both δ-aminolevulinic acid (ALA) and Hb synthesis. In contrast, addition of hemin elevated the levels of all iron species as well as the Hb synthesis but reduced the TfR expression and ALA contents in both butyrate treated and untreated cells. These results suggest that Hb synthesis is controlled by TfR expression, and that the ALA synthesis is suppressed by iron released from heme and/or Hb due to lowered expression of TfR.     

Use of high-resolution magic angle spinning nuclear magnetic resonance spectroscopy for in situ studies of low-molecular-mass compounds in red algae

The use of high-resolution magic angle spinning NMR spectroscopy for in situ studies of low-molecular-mass compounds in red algae has been studied. The impact of different acquisition parameters on the resulting T(2)-filtered one-dimensional high-resolution magic angle spinning (1)H NMR spectra is described. The technique was used for in situ identification and quantification of some low-molecular-mass algal metabolites.     

Cepabactin from Pseudomonas cepacia, a new type of siderophore

In iron-deficient conditions of growth Pseudomonas cepacia ATCC 25416 excreted both pyochelin and a low-molecular-mass compound which strongly chelated iron(III), and facilitated iron translocation as demonstrated by growth and uptake experiments. The name cepabactin is proposed for this new siderophore. Comparisons of UV-visible spectra and chromatographic behaviour, together with 1H-NMR spectra, led to the conclusion that cepabactin is 1-hydroxy-5-methoxy-6-methyl-2(1H)-pyridinone, a compound which can be considered as a cyclic hydroxamate, but also as a heterocyclic analogue of catechol. This pyridinone has already been described by other workers as an antibiotic produced by Pseudomonas alcaligenes, and by a soil isolate closely related to Pseudomonas cepacia. Thus, cepabactin appears to act as a siderophore for more than one species of non-fluorescent pseudomonad.     

Non-transferrin-bound iron in plasma or serum from patients with idiopathic hemochromatosis. Characterization by high performance liquid chromatography and nuclear magnetic resonance spectroscopy

The nature of non-transferrin-bound iron in the plasma or serum of iron-overloaded hemochromatosis patients was studied by high performance liquid chromatography (HPLC) and high resolution nuclear magnetic resonance (NMR). 500-MHz proton Hahn spin-echo NMR spectra of plasma or serum, combined with the use of the iron chelator desferrioxamine, suggests complexation of iron ions with citrate and a possible involvement of acetate. Addition of FeCl3 to hemochromatosis samples broadened the NMR signals from citrate. HPLC analysis rigorously confirmed the presence of an iron-citrate complex in ultrafiltrates of plasma or serum studies with added FeCl3 or desferrioxamine supported this conclusion. It is proposed that non-transferrin-bound iron in the plasma of iron-overloaded patients exists largely as complexes with citrate and possibly also as ternary iron-citrate-acetate complexes. The presence of such complexes would account for the ability of non-transferrin-bound iron to be measurable by the bleomycin assay and for its rapid clearance from the circulation by the liver.     

Preparation of low-molecular-mass peptides by enzyme autolysis oF Paphia undulata

Paphia undulata was used to prepare low-molecular-mass peptides by enzyme autolysis. The optimum condition for autolysis was studied to reach a maximum yield of peptides. Results showed that the yield of low-molecular-mass peptides was 4.33% and 78.57% of the total protein can be recovered under the optimized condition: pH = 6.5, temperature = 45 °C, reaction time = 4 h, ratio of starting material and water = 1:3 and the enzyme dosage was 1374.0 U/g.     

Polymyxin B inhibits insulin-induced glucose transporter and IGF II receptor translocation in isolated adipocytes.

In isolated adipocytes, polymyxin B inhibited insulin-induced glucose incorporation into lipids in a dose-dependent manner, while polymyxin E, a structurally related antibiotic, was ineffective. To approach the mechanism of this effect, the subcellular distribution of the glucose transporter Glut 4 was investigated. Adipocytes were pretreated without or with polymyxin B before insulin stimulation, subcellular fractionation was performed and Glut 4 was detected by immunodetection. Incubation of adipocytes with polymyxin B prevented the insulin-induced appearance of Glut 4 in the plasma membranes, but did not prevent their decrease from the low-density microsomal fraction. A lower purity of the plasma membrane fractions, a detergent effect of polymyxin B on the membranes or an interference of the substance with the immunodetection of the Glut 4 molecules were excluded. These results suggest that polymyxin B was interfering with the Glut 4 translocation process stimulated by insulin in adipocytes. In a similar fashion, polymyxin B inhibited the insulin-induced increase in IGF II binding to adipocytes. This resulted from a blockade of the appearance of IGF II receptors in the plasma membranes. Since low-molecular-mass GTP-binding proteins have been implicated in the regulation of vesicular trafficking, we have used [alpha-32P]GTP binding to analyze such proteins in adipocyte fractions, after SDS/PAGE and transfer to nitrocellulose. Specific and distinct subsets of GTP-binding proteins were revealed in plasma membrane and low-density microsomal fractions of control adipocytes, whether they were stimulated or not with insulin. Polymyxin B treatment of adipocytes markedly modified the profile of the low-molecular-mass GTP-binding proteins in plasma membranes, but not in low-density microsomal fractions. Our results suggest that polymyxin B was interfering with the exocytotic process of the Glut 4 and IGF II receptor-containing vesicles, perhaps at the fusion step between vesicles and plasma membranes.     

Antioxidant protection by haemopexin of haem-stimulated lipid peroxidation.

Haem (ferrous protoporphyrin IX) is a reactive low-molecular-mass form of iron able to participate in oxygen-radical reactions that can lead to the degradation of proteins, lipids, carbohydrates and DNA. Oxygen-radical reactions are likely to occur upon tissue damage. Extracellular fluids rely on antioxidant mechanisms different from those found inside the cell, and circulating proteins limit radical reactions by converting pro-oxidant forms of iron into less-reactive forms. Of the compounds tested, only apohaemopexin and the chain-breaking antioxidant butylated hydroxytoluene inhibited (by more than 90%) haemin-stimulated peroxidation as measured by formation of conjugated dienes, thiobarbituric acid-reactive material from linolenic acid or peroxidation-induced phospholipid fluorescence. Haptoglobin, the haemoglobin-binding serum protein, was ineffective. Conversely, only haptoglobin significantly inhibited haemoglobin-stimulated lipid peroxidation. Iron-salt-induced lipid peroxidation was inhibited only by apotransferrin and the iron-chelator desferrioxamine. All lipid peroxidations were inhibited by the radical scavengers butylated hydroxytoluene and propyl gallate. These findings support the concept that transport and conservation of body iron stores are closely linked to antioxidant protection.     

Plasma ferritin and other parameters related to iron metabolism in piglets

The evolution of mean plasma ferritin values, hematocrit, hemoglobin, plasma iron concentration and total plasma iron binding capacity were studied during the growth of piglets from 0 to 50 days. The results obtained point to a massive mobilization of iron from storage sites during the second and third weeks of life of these animals. Apart from plasma ferritin values and the total plasma iron binding capacity, the coefficient of utilization may be considered as another parameter to be taken into account upon evaluating iron deposits in piglets.     

Development of a pharmaceutical apotransferrin product for iron binding therapy.

High-dose chemotherapy of patients with haematological malignancies results in extracellular iron accumulation and appearance of non-transferrin-bound iron, which is thought to predispose the patients to septic infections and contribute to organ toxicity. We describe the development of a human plasma-derived apotransferrin product for iron binding therapy. The product is purified from Cohn fraction IV of human plasma by two ion exchange chromatography steps and ultrafiltration. The process comprises solvent detergent treatment as the main virus inactivation step and 15 nm virus filtration and polyethylene glycol precipitation as removal steps for physico-chemically resistant infectious agents. Product characterization by electrospray and MALDI-TOF mass spectrometry indicated no other chemical modifications than N-linked glycan chains and disulphide bonds, except minor oxidation. The purity of the product was more than 98%, main impurities being IgG, IgA and hemopexin. The product had intact iron binding capacity and native conformation. A stable liquid formulation for the finished product was developed. The product has proved safe and well tolerated in early clinical trials in iron binding therapy.     

Depot iron in the rat liver after hypertransfusion with rat erythrocytes

In the rat liver the deposition of iron was measured after hypertransfusion with rat erythrocytes. The liver iron fractions were studied during four weeks after the hypertransfusions. In the first week the haemosiderin iron fraction increased together with the ferritin iron fraction. Most iron was deposited as ferritin iron. In the last week of the experiments, while the ferritin iron fraction still increased, the haemosiderin iron fraction decreased. At the same time plasma iron was utilized when erythropoiesis, which had been suppressed by the hypertransfusion, recommenced. It is suggest that, under these experimental conditions, liver haemosiderin iron is used in haemoglobin synthesis.     

Hierro e infección fúngica invasiva

Iron is an essential factor for both the growth and virulence of most of microorganisms. As a part of the innate (or nutritional) immune system, mammals have developed different mechanisms to store and transport this element in order to limit free iron bioavailability. To survive in this hostile environment, pathogenic fungi have specific uptake systems for host iron sources, one of the most important of which is based on the synthesis of siderophores-soluble, low-molecular-mass, high-affinity iron chelators. The increase in free iron that results from iron-overload conditions is a well-established risk factor for invasive fungal infection (IFI) such as mucormycosis or aspergillosis. Therefore, iron chelation may be an appealing therapeutic option for these infections. Nevertheless, deferoxamine –the first approved iron chelator– paradoxically increases the incidence of IFI, as it serves as a xeno-siderophore to Mucorales. On the contrary, the new oral iron chelators (deferiprone and deferasirox) have shown to exert a deleterious effect on fungal growth both in vitro and in animal models. The present review focuses on the role of iron metabolism in the pathogenesis of IFI and summarises the preclinical data, as well as the limited clinical experience so far, in the use of new iron chelators as treatment for mucormycosis and invasive aspergillosis.     

Suppressive effect of deferoxamine on the growth of Pneumocystis carinii in vitro.

The effects of the iron chelator deferoxamine on the growth of rat-derived Pneumocystis carinii in culture with human embryonic lung fibroblasts were studied. Growth inhibition was calculated by comparison of trophozoite numbers in replicate samples of supernatant of treated and untreated samples. Deferoxamine, in concentrations safely achievable in humans (5-15 micrograms/ml, corresponding to 7.6-22.8 microM), reproducibly suppressed P. carinii growth in a dose-dependent manner. The suppressive effect was reversed by prior iron saturation of the deferoxamine. Since the utility of current therapeutic agents for P. carinii disease is limited by toxicity and incomplete efficacy, the role of iron chelation as an adjunct to anti-Pneumocystis chemotherapy merits further investigation.     

Melatonin controls oxidative stress and modulates iron, ferritin, and transferrin levels in adriamycin treated rats

AIM: Chemotherapy with adriamycin (ADR) is limited by its iron-mediated pro-oxidant toxicity. Because melatonin (MLT) is a broad spectrum antioxidant, we investigated the ability of MLT to control iron, its binding proteins, and the oxidative damage induced by ADR. MAIN METHODS: ADR was given as single i.p. dose of 10 mg kg(-1) body weight into male rats. MLT at a dose of 15 mg kg(-1) was injected daily for 5 days before ADR treatment followed by another injection for 5 days. Biochemical methods were used for this investigation. KEY FINDINGS: ADR injection caused elevations in plasma creatine kinase isoenzyme, lactic dehydrogenase, and aminotransferases, iron, ferritin, and transferrin. These changes were associated with increases in lipid peroxidation and protein oxidation as well as decreases in glutathione (GSH) levels and glutathione-S-transferase (GST) activity, while glutathione peroxidase (GSH-Px), and catalase (CAT) activity were elevated in the heart and liver of ADR treated rats. In the MLT+ADR group, the cardiac and hepatic function parameters and the levels of iron, transferrin and ferritin in plasma were normalized to control levels. The rats that were subjected to MLT+ADR had normalized CAT and GSH-Px activity and decreased TBARS and protein carbonyl levels compared the group only treated with ADR. GST activity and GSH concentration in the heart and liver were normalized when MLT accompanied ADR treatment. SIGNIFICANCE: MLT ameliorated oxidative stress by controlling iron, and binding protein levels in ADR treated rats demonstrating the usefulness of adriamycin in cancer chemotherapy and allowing a better management of iron levels.     

Characterization of non-transferrin-bound iron clearance by rat liver

Recent evidence suggests that the hepatic iron-loading characteristic of hemochromatosis may result in part from efficient hepatic clearance of non-transferrin-bound iron, which is increased in this disorder. However, this hypothesis assumes that hepatic clearance remains highly efficient despite excess iron stores. We therefore studied hepatic uptake of non-transferrin-bound iron in the single-pass perfused rat liver under varying conditions. Animals were iron loaded or depleted by dietary manipulation, but no changes in the efficiency of ferrous iron uptake or the kinetic parameters were seen (single-pass extraction, 59-74%; Km, 16-19 microM; Vmax, 30-32 nmol X min-1 X g liver-1). Added divalent zinc, cobalt, and manganese ions reversibly inhibited ferrous iron uptake and the inhibition by zinc was shown to be competitive. Uptake required calcium, was markedly temperature-sensitive (delta E = 14.3 Kcal/mol), and was relatively insensitive to inhibition of cellular energy metabolism. Particles consistent with ferritin cores were seen in lysosomes of hepatic parenchymal cells within 30 min of perfusion with ferrous iron. These results suggest that ferrous iron is cleared from plasma by a passive, saturable transport process that is not regulated by the iron content of the liver and that may be shared with other transition metal ions. Because clearance is highly efficient, increased levels of non-transferrin-bound iron in plasma may present the liver with an obligatory iron load resulting in progressive accumulation and toxicity.     

SidL, an Aspergillus fumigatus transacetylase involved in biosynthesis of the siderophores ferricrocin and hydroxyferricrocin

The opportunistic fungal pathogen Aspergillus fumigatus produces four types of siderophores, low-molecular-mass iron chelators: it excretes fusarinine C (FsC) and triacetylfusarinine C (TAFC) for iron uptake and accumulates ferricrocin (FC) for hyphal and hydroxyferricrocin (HFC) for conidial iron distribution and storage. Siderophore biosynthesis has recently been shown to be crucial for fungal virulence. Here we identified a new component of the fungal siderophore biosynthetic machinery: AFUA_1G04450, termed SidL. SidL is conserved only in siderophore-producing ascomycetes and shows similarity to transacylases involved in bacterial siderophore biosynthesis and the N(5)-hydroxyornithine:anhydromevalonyl coenzyme A-N(5)-transacylase SidF, which is essential for TAFC biosynthesis. Inactivation of SidL in A. fumigatus decreased FC biosynthesis during iron starvation and completely blocked FC biosynthesis during iron-replete growth. In agreement with these findings, SidL deficiency blocked conidial accumulation of FC-derived HFC under iron-replete conditions, which delayed germination and decreased the size of conidia and their resistance to oxidative stress. Remarkably, the sidL gene is not clustered with other siderophore-biosynthetic genes, and its expression is not affected by iron availability. Tagging of SidL with enhanced green fluorescent protein suggested a cytosolic localization of the FC-biosynthetic machinery. Taken together, these data suggest that SidL is a constitutively active N(5)-hydroxyornithine-acetylase required for FC biosynthesis, in particular under iron-replete conditions. Moreover, this study revealed the unexpected complexity of siderophore biosynthesis, indicating the existence of an additional, iron-repressed N(5)-hydroxyornithine-acetylase.     

Plasma chemical composition in the goosefish (Lophius piscatorius, L)

124 specimens of Lophius piscatorius , L. have been studied (22 males, 90 immature females and 12 sexually mature females) from the Mediterranean coast between October 1968 and February 1971. The plasma values of total proteins, Cl^-, K⁺, Na⁺, plasma iron, total iron binding capacity and plasma copper have been determined. These values are related to weight, length, sex and sexual maturity, depth of trawl and season of the year. The electrolyte content is somewhat lower than given for this species. A clear seasonal variation is observed, with a decrease in the plasma electrolyte. The iron and copper plasma and TIBC values agree with the few values previously recorded and it is thought that they will be of interest due to the scarcity of data on fish.