M?ssbauer spectroscopy, electron microscopy and electron diffraction studies of the iron cores in various human and animal haemosiderins

M?ssbauer spectroscopy has indicated significant differences in the iron-containing cores of various haemosiderins. In the present study, haemosiderin was isolated from a number of animal species including man. In addition, haemosiderin was isolated from patients with primary idiopathic haemochromatosis or with secondary (transfusional) iron-overload. The iron cores of the animal and normal human haemosiderin appear to be very similar by M?ssbauer spectroscopy, and the electron diffraction data indicate a ferrihydrite structure similar to that of ferritin cores. The haemosiderin isolated from secondary iron-overload shows anomalous behaviour in its temperature-dependent M?ssbauer spectra. This can be understood in terms of the microcrystalline goethite structure of the cores as indicated by electron diffraction. The haemosiderin cores obtained in the case of primary haemochromatosis have an amorphous Fe(III) oxide structure and show M?ssbauer spectra characteristic of a magnetically disordered material, which only orders at very low temperatures.     

Biochemical studies of the iron cores and polypeptide shells of haemosiderin isolated from patients with primary or secondary haemochromatosis

Haemosiderin isolated from different iron-loading syndromes, primary haemochromatosis (PHC) and secondary haemochromatosis (SHC) biochemically exhibited differences in both their iron core and peptide composition. The rate of release of iron from PHC haemosiderin to oxalate was 3-fold greater than that from SHC haemosiderin. The major peptides separated by SDS-PAGE showed a major band at Mr 20,000 for PHC haemosiderin and at Mr 15,000 for SHC haemosiderin.     

Differences in the pattern of iron accumulation in primary and secondary hemochromatosis. Diagnostic importance and clinical consequences]

In secondary haemochromatosis up to fourfold higher amounts of iron are tolerated in the organism than in primary (hereditary) haemochromatosis. This is connected with the marked iron storage of macrophages in secondary iron overloading, which is relatively without any dangers. In primary haemochromatosis, however, a relative insufficiency of storage of extrahepatic macrophages can be observed for iron, a fact which favours a premature parenchymatous iron storage leading to organ lesions. Because of the discrepant behaviour of macrophages characteristic, diagnostically relevant differences will occur in the pattern of iron storage in the bone-marrow, spleen and small intestine between primary and secondary haemochromatosis.     

Erythrocyte ferritin content in idiopathic haemochromatosis and alcoholic liver disease with iron overload.

The erythrocyte ferritin content was measured in patients with either idiopathic haemochromatosis or alcoholic liver disease and iron overload to define its value as a marker for an excess of tissue iron. The mean erythrocyte ferritin content in patients with untreated idiopathic haemochromatosis was increased 60-fold and fell with phlebotomy. After phlebotomy many patients had an increased red cell ferritin content despite normal serum ferritin concentrations. That this reflected persistent iron overload with inadequate phlebotomy was suggested by the higher serum iron concentrations, percentage transferrin saturation, and urinary excretion of iron after administration of desferrioxamine, together with a lower annual iron loss by phlebotomy in this group compared with patients with treated disease and normal red cell ferritin content. The mean erythrocyte ferritin content in patients with alcoholic liver disease and iron overload was increased only sevenfold, and the ratio of erythrocyte to serum ferritin clearly discriminated these patients from those with idiopathic haemochromatosis. The determination of erythrocyte ferritin content is a useful non-invasive test for diagnosing idiopathic haemochromatosis, monitoring the effect of phlebotomy in this disorder, and distinguishing patients with this disorder from those with alcoholic liver disease with iron overload.     

Iron K-edge absorption spectroscopic investigations of the cores of ferritin and haemosiderins.

The extended X-ray absorption fine structure (EXAFS) associated with the iron K-edge has been measured and interpreted for ferritin and haemosiderin extracted from horse spleen, and haemosiderin extracted from the livers of humans with treated primary haemochromatosis, and from the spleens of humans with treated secondary haemochromatosis. For ferritin, the data are consistent with, on average, each iron atom being in an environment comprised of approx. six oxygen atoms at 1.93 +/- 0.02 A, approx. 1.5 iron atoms at 2.95 +/- 0.02 A and approx. 1.1 iron atoms at 3.39 +/- 0.02 A, with a further shell of oxygens at approx. 3.6 A. Iron in horse spleen haemosiderin is in an essentially identical local environment to that in horse spleen ferritin. In contrast, the EXAFS data for primary haemochromatosis haemosiderin indicate that the iron-oxide core is amorphous; only a single shell of approx. six oxygen atoms at approx. 1.94 +/- 0.02 A being apparent. Secondary haemochromatosis haemosiderin shows an ordered structure with approx. 1.4 iron atoms at both 2.97 +/- 0.02 and 3.34 +/- 0.02 A. This arrangement of iron atoms is similar to that in horse spleen haemosiderin, but the first oxygen shell is split with approx. 2.9 atoms at 1.90 +/- 0.02 A and approx. 2.7 at 2.03 +/- 0.02 A, indicative of substantial structural differences between secondary haemochromatosis haemosiderin and horse spleen haemosiderin.     

Biochemical and biophysical investigations of the ferrocene-iron-loaded rat. An animal model of primary haemochromatosis.

Male Wistar rats fed with ferrocene had high hepatic iron loading (7.24 +/- 1.97 mg Fe/g tissue) after 6 weeks, principally located in lysosomes, which was comparable to the levels and distribution determined in human haemochromatosis. The two iron-storage proteins, ferritin and haemosiderin were isolated from the livers of the ferrocene-loaded rats and their iron cores were investigated by M?ssbauer spectroscopy and inductively coupled plasma-emission spectrometry. Ferrihydrite was the predominant form of iron present in both ferritin and haemosiderin, while haemosiderin contained higher amounts of phosphorus, magnesium, calcium and barium, then either normal or ferrocene-loaded ferritin. Free-radical-mediated damage in the iron-loaded livers was inferred by the significant depletion of alpha-tocopherol in both the livers and subcellular hepatic lysosomal fraction, which inversely correlated with the increasing iron content (r = -0.61; P less than 0.05) and was associated with increased fragility of the lysosomal membranes.     

Changes in function of iron-loaded alveolar macrophages after in vivo administration of desferrioxamine and/or chloroquine

Both desferrioxamine (DFO) and chloroquine can significantly reduce hepatic iron in experimental animals with iron overload by chelating iron from the low-molecular-weight pool or decreasing iron uptake by the transferrin-transferrin receptor cycle, respectively. However, no previous studies have investigated whether combination therapy of these two drugs would further decrease the tissue iron overload as well as iron-induced toxicity. Chloroquine administration, 15 mg/kg, 5x/week, to rats during the iron loading regime, 10 mg/kg, 3x/week for 4 weeks, significantly decreased both hepatic (54%) and macrophage iron content (24%). However when administered in combination with desferrioxamine, 10 mg/kg, 3x/week for 2 weeks at the cessation of iron loading, no further reduction of hepatic iron content was noted while the iron content of the macrophages significantly increased, possibly indicating the flux of ferrioxamine through these cells. Further studies are warranted to investigate the speciation of iron within these macrophages. Macrophages isolated from chloroquine-treated iron loaded rats showed a reduction in latent NFkappaB activation and a significant increase in lipopolysaccharide-stimulated nitrite release by comparison to these parameters in iron loaded macrophages. Co-administration of chloroquine and desferrioxamine normalised the latent activity of NFkappaB to that of control macrophages as well as increasing LPS-stimulated NO release towards control values. However, DFO alone did not have any significant effect upon either of these parameters. Such results may have important relevance for the reduced immune function of iron loaded macrophages isolated from thalassaemia patients receiving chelation therapy and their propensity to increased infection.     

Quantitative magnetic analysis reveals ferritin-like iron as the most predominant iron-containing species in the murine Hfe-haemochromatosis

Quantitative analysis of the temperature dependent AC magnetic susceptibility of freeze-dried mouse tissues from an Hfe hereditary haemochromatosis disease model indicates that iron predominantly appears biomineralised, like in the ferritin cores, in the liver, the spleen and duodenum. The distribution of the amount of ferritin-like iron between genders and genotypes coincides with that of elemental iron and nonheme iron. Importantly, the so-called paramagnetic iron, a quantity also determined from the magnetic data and indicative of nonmineralised iron forms, appears only marginally increased when iron overload takes place.     

Evolutionary inheritance of elemental stoichiometry in phytoplankton

The elemental composition of phytoplankton is a fusion of the evolutionary history of the host and plastid, resulting in differences in genetic constraints and selection pressures associated with environmental conditions. The evolutionary inheritance hypothesis predicts similarities in elemental composition within related taxonomic lineages of phytoplankton. To test this hypothesis, we measured the elemental composition (C, N, P, S, K, Mg, Ca, Sr, Fe, Mn, Zn, Cu, Co, Cd and Mo) of 14 phytoplankton species and combined these with published data from 15 more species from both marine and freshwater environments grown under nutrient-replete conditions. The largest differences in the elemental profiles of the species distinguish between the prokaryotic Cyanophyta and primary endosymbiotic events that resulted in the green and red plastid lineages. Smaller differences in trace element stoichiometry within the red and green plastid lineages are consistent with changes in trace elemental stoichiometry owing to the processes associated with secondary endosymbioses and inheritance by descent with modification.     

Computed tomography for determining liver iron content in primary haemochromatosis.

Dual-energy computed tomography (CT) was used to estimate hepatic iron concentration in eight patients with primary haemochromatosis with varying degrees of iron overload. The values corresponded closely with these derived from chemical analysis of liver tissue obtained by biopsy (correlation coefficient 0.993). Dual-energy CT therefore seems to provide an accurate and non-invasive alternative to liver biopsy as a means of measuring liver iron concentration in patients with primary haemochromatosis and possibly other iron overload states.     

Effects of copper and ceruloplasmin on iron transport in the Caco 2 cell intestinal model

Previous studies have implicated copper proteins, including ceruloplasmin, in intestinal iron transport. Polarized Caco2 cells with tight junctions were used to examine the possibilities that (a) ceruloplasmin promotes iron absorption by enhancing release at the basolateral cell surface and (b) copper deficiency reduces intestinal iron transport. Iron uptake and overall transport were followed for 90 min with 1 &mgr;M 59Fe(II) applied to the apical surface of Caco2 cell monolayers. Apotransferrin (38 &mgr;M) was in the basolateral chamber. Induction of iron deficiency with desferrioxamine (100 &mgr;M; 18 h) markedly increased uptake and overall transport of iron. Uptake increased from about 20% to about 65% of dose, and overall 59Fe transport from <1% to 60% of dose. On the basis of actual iron released into the basal chamber (measured with bathophenanthroline), transport increased 8-fold. Desferrioxamine pretreatment reduced cellular Fe by 55%. The addition of freshly isolated, enzymatically active human ceruloplasmin to the basolateral chamber during absorption had no effect on uptake or transport of iron by the cells. Unexpectedly, pretreatment with three different chelators of copper (18 h), which reduced cellular levels about 40%, more than doubled iron uptake and raised overall transport to 20%. This was so, whether or not cells were also made iron deficient with desferrioxamine. Acute addition of 1 &mgr;M Cu(II) to the apical chamber had no significant effect upon iron uptake, retention, or transport in iron deficient or normal cells, in the presence of absence of ascorbate. We conclude that intestinal absorption of Fe(II) is unlikely to depend upon plasma ceruloplasmin, and that cuproproteins involved in this form of iron transport must be binding copper tightly.     

Comparison of cytosolic products formed in rat liver in response to parenteral and dietary iron loading

Two different methods were used to create a situation of iron (Fe) overload in rats. One group of rats received Fe dextran, and another group of rats received a carbonyl Fe-enriched diet. The ferritins present in the liver cytosol of these rats were isolated and compared. From each group, two cytosolic products were isolated with the use of ultracentrifugation: a cytosolic ferritin fraction (CF) and a (slower sedimenting) light ferritin fraction (CLF). There were no differences with respect to the protein coat (subunit composition and amino acid analysis). Analysis of the Fe core revealed that the two CF fractions were similar, whereas the two CLF fractions differed with respect to their Fe content and to the packing of their cores. The carbonyl CLF product contained less Fe atoms/molecule, which, moreover, seemed to be packed in a less compact way.     

Haemosiderin and tissue damage

High levels of haemosiderin occur in iron overload syndromes such as idiopathic haemochromatosis or secondary iron overload in thalassaemic patients; haemosiderin is the predominant iron-storage compound in such cases. It consists of a large aggregate of FeOOH cores, many of which have an incomplete shell of protein, and is probably derived from ferritin by lysosomal proteolysis. In addition, some chemical degradation of the ferritin cores appears to occur on conversion to haemosiderin. Other biochemical components are phosphate and magnesium, which may be adsorbed to the core surface, and perhaps certain lipids. Haemosiderin may have a central role, either directly or indirectly, in iron cytotoxicity and therefore the chemistry and biochemistry of this material warrants further study.     

Hepatic iron deposition in human disease and animal models

Iron deposition occurs in parenchymal cells of the liver in two major defects in human subjects (i) in primary iron overload (genetic haemochromatosis) and (ii) secondary to anaemias in which erythropolesis is increased (thalassaemia). Transfusional iron overload results in excessive storage primarily in cells of the reticule endothelial system. The storage patterns in these situations are quite characteristic. Excessive iron storage, particularly in parenchymal cells eventually results in fibrosis and cirrhosis. There is no animal model or iron overload which completely mimics genetics haemochromatosis but dietary iron loading with carbonyl iron or ferrocene does produce excessive parenchymal iron stores in the rat. Such models have been used to study iron toxicity and the action of iron chelators in the effective removal of excessive iron stores.     

M?ssbauer spectroscopic studies of the cores of human, limpet and bacterial ferritins

Ferritin cores from human spleen, limpet (Patella vulgata) haemolymph and bacterial (Pseudomonas aeruginosa) cells have been investigated using 57Fe M?ssbauer spectroscopy. The M?ssbauer spectra were recorded over a range of temperatures from 1.3 to 78 K, all the spectra are quadrupole-split doublets with similar quadrupole splittings and isomer shifts, characteristic of iron(III), while at sufficiently low temperatures the spectra of all the samples show well-resolved magnetic splitting. At intermediate temperatures, the spectra from the human ferritin exhibit typical superparamagnetic behaviour, while those from the bacterial ferritin show behaviour corresponding to a transition from a magnetically ordered to a paramagnetic state. The spectra from the limpet ferritin show a complex combination of the two effects. The results are discussed in terms of the magnetic behaviour of small particles. The data are consistent with magnetic ordering temperatures of about 3 and 30 K for the bacterial and limpet ferritin cores, respectively, while the data indicate that the magnetic ordering temperature for the human ferritin cores must be above 50 K. These differences are interpreted as being related to different densities of iron in the cores and to variations in the composition of the cores. The human ferritin cores are observed to have a mean superparamagnetic blocking temperature of about 40 K, while that of the limpet ferritin cores is about 25 K. This difference is interpreted as being due not only to different mean numbers of iron atoms in the two types of core but also to the higher degree of crystallinity in the cores of the human ferritin.     

Ferrous ion autoxidation and its chelation in iron-loaded human liver HepG2 cells.

Ferrous ion (Fe(2+)) is long thought to be the most likely active species, producing oxidants through interaction of Fe(2+) with oxygen (O(2)). Because current iron overload therapy uses only Fe(3+) chelators, such as desferrioxamine (DFO), we have tested a hypothesis that addition of a Fe(2+) chelator, 2,2'-dipyridyl (DP), may be more efficient and effective in preventing iron-induced oxidative damage in human liver HepG2 cells than DFO alone. Using ferrozine as an assay for iron measurement, levels of cellular iron in HepG2 cells treated with iron compounds correlated well with the extent of lipid peroxidation (r = 0.99 after log transformation). DP or DFO alone decreased levels of iron and lipid peroxidation in cells treated with iron. DFO + DP together had the most significant effect in preventing cells from lipid peroxidation but not as effective in decreasing overall iron levels in the cells. Using ESR spin trapping technique, we further tested factors that can affect oxidant-producing activity of Fe(2+) with dissolved O(2) in a cell-free system. Oxidant formation enhanced with increasing Fe(2+) concentrations and reached a maximum at 5 mM of Fe(2+). When the concentration of Fe(2+) was increased to 50 mM, the oxidant-producing activity of Fe(2+) sharply decreased to zero. The initial ratio of Fe(3+):Fe(2+) did not affect the oxidant producing activity of Fe(2+). However, an acidic pH (< 3.5) significantly slowed down the rate of the reaction. Our results suggest that reaction of Fe(2+) with O(2) is an important one for oxidant formation in biological system, and therefore, drugs capable of inhibiting redox activity of Fe(2+) should be considered in combination with a Fe(3+) chelator for iron overload chelation therapy.     

Structure and composition of ferritin cores isolated from human spleen, limpet (Patella vulgata) hemolymph and bacterial (Pseudomonas aeruginosa) cells

Ferritin cores isolated from human spleen, limpet (Patella vulgata) hemolymph and bacterial (Pseudomonas aeruginosa) cells have been investigated by high resolution transmission electron microscopy, electron diffraction and chemical analysis. Hemosiderin particles isolated from thalassemic spleens also have been studied. The results show that there is a marked difference in structure and composition of the biomineral phases. Human ferritin and hemosiderin particles are single domain crystals of hydrated iron (III) oxide (ferrihydrite). Lattice fringes were low in contrast and often discontinuous within the central regions of the core. Heat treatment of human ferritins results in a 5 A shrinkage in particle size and an increase in the single crystalline nature of the core. In contrast, lattice images and electron diffraction of limpet and bacterial cores show no evidence of long-range crystallographic order. Chemical analysis indicates a high inorganic phosphate (Pi) (Fe/Pi = 1.71) content in bacterial ferritin compared with human ferritin (thalassemic) (Fe/Pi = 21.0). The high Pi content of bacterial ferritin suggests a hydrated amorphous iron (III) phosphate mineral core. Structural disorder within the limpet and bacterial cores may be associated with increased Pi content and increased oxidation in Fe(II), resulting in rapid mineral deposition. Growth of the iron (III) oxide cores in human ferritin is discussed on the basis of high resolution electron microscopy results.     

Development of potential iron chelators for the treatment of Friedreich's ataxia: ligands that mobilize mitochondrial iron

Friedreich's ataxia (FA) is a crippling neurodegenerative disease that is due to iron (Fe) overload within the mitochondrion. One therapeutic intervention may be the development of a chelator that could remove mitochondrial Fe. We have implemented the only well characterized model of mammalian mitochondrial Fe overload to examine the Fe chelation efficacy of novel chelators of the 2-pyridylcarboxaldehyde isonicotinoyl hydrazone (PCIH) class. In this model we utilize reticulocytes treated with the haem synthesis inhibitor succinylacetone which results in mitochondrial Fe-loading. Our experiments demonstrate that in contrast to desferrioxamine, several of the PCIH analogues show very high activity at mobilizing (59)Fe from (59)Fe-loaded reticulocytes. Further studies on these ligands in animals are clearly warranted considering their potential to treat FA.     

Measuring "free" iron levels in Caenorhabditis elegans using low-temperature Fe(III) electron paramagnetic resonance spectroscopy

Oxidative stress, caused by free radicals within the body, has been associated with the process of aging and many human diseases. Because free radicals, in particular superoxide, are difficult to measure, an alternative indirect method for measuring oxidative stress levels has been used successfully in Escherichia coli and yeast. This method is based on a proposed connection between elevated superoxide levels and release of iron from solvent-exposed [4Fe-4S] enzyme clusters that eventually leads to an increase in hydroxyl radical production. In past studies using bacteria and yeast, a positive correlation was found between superoxide production or oxidative stress due to superoxide within the organism and electron paramagnetic resonance (EPR) detectable "free" iron levels. In the current study, we have developed a reliable and efficient method for measuring "free" iron levels in Caenorhabditis elegans using low-temperature Fe(III) EPR at g=4.3. This method uses synchronized worm cultures grown on plates that are homogenized and treated with desferrioxamine, an Fe(III) chelator, prior to packing the EPR tube. Homogenization was found not to alter "free" iron levels, whereas desferrioxamine treatment significantly raised these levels, indicating the presence of both Fe(II) and Fe(III) in the "free" iron pool. The correlation between free radical levels and the observed "free" iron levels was examined by using heat stress and paraquat treatment. The intensity of the Fe(III) EPR signal, and thus the concentration of the "free" iron pool, varied with the treatments that altered radical levels without changing the total iron levels. This study provides the groundwork needed to uncover the correlation among oxidative stress, "free" iron levels, and longevity in C. elegans.     

Interaction between iron(II) and hydroxamic acids: oxidation of iron(II) to iron(III) by desferrioxamine B under anaerobic conditions

Interaction between iron(II) and acetohydroxamic acid (Aha), alpha-alaninehydroxamic acid (alpha-Alaha), beta-alaninehydroxamic acid (beta-Alaha), hexanedioic acid bis(3-hydroxycarbamoyl-methyl)amide (Dha) or desferrioxamine B (DFB) under anaerobic conditions was studied by pH-metric and UV-Visible spectrophotometric methods. The stability constants of complexes formed with Aha, alpha-Alaha, beta-Alaha and Dha were calculated and turned out to be much lower than those of the corresponding iron(II) complexes. Stability constants of the iron(II)-hydroxamate complexes are compared with those of other divalent 3d-block metal ions and the Irving-Williams series of stabilities was found to be observed. Above pH 4, in the reactions between iron(II) and desferrioxamine B, the oxidation of the metal ion to iron(III) by the ligand was found. The overall reaction that resulted in the formation of the tris-hydroxamato complex [Fe(HDFB)]+ and monoamide derivative of DFB at pH 6 is: 2Fe2+ + 3H4DFB+ = 2[Fe(HDFB)]+ + H3DFB-monoamide+ + H2O + 4H+. Based on these results, the conclusion is that desferrioxamine B can uptake iron in iron(III) form under anaerobic conditions.