Resonance Raman scattering on the haem group of cytochrome c

Resonance Raman spectra of the haem group of 8 × 10^?5 M horse heart ferro- and ferricytochrome c solutions have been obtained. The spectra are almost identical to that of haemoglobin. The frequency of the Raman line near 1370 cm^?1, which in haemoglobin is sensitive to the position of the haem iron, indicates that the iron atom of cytochrome c lies in the plane of the porphyrin for both oxidation states.     

A Simple and Rapid Method for the Determination of "free" Iron in Biological Fluids

We present a convenient method for determining "free" or non-protein-bound iron in biological fluids. The new method is based on the bathophenantroline method for determination of total serum iron, and comprises binding of iron by a chromogenic chelator (bathophenantroline-disulphonate, BPS), which is specific for ferrous iron. The ferrous complex of BPS absorbs strongly at 535 nm, and the detection limit is less than 1 &#119 M in a sample size of 50 &#119 l. The chelator does not liberate iron from either haemoglobin or transferrin. Interference from copper or zinc in concentrations up to 50 &#119 M does not significantly disturb measurements. The main problem when measuring in blood plasma, the high and fluctuating background in the region around 535 nm, has been overcome through filtering techniques. Data from measurements of ferrous iron in microdialysate, cerebrospinal fluid, and blood plasma in different animal models and clinical conditions are presented as illustrative examples of the usefulness of the method. The method allows the determination of ferric, as well as ferrous, iron in the same sample.     

Iron incorporation and post-malaria anaemia

Background

Iron supplementation is employed to treat post-malarial anaemia in environments where iron deficiency is common. Malaria induces an intense inflammatory reaction that stalls reticulo-endothelial macrophagal iron recycling from haemolysed red blood cells and inhibits oral iron absorption, but the magnitude and duration of these effects are unclear.

Methodology/Principal Findings

We examined the red blood cell incorporation of oral administered stable isotopes of iron and compared incorporation between age matched 18 to 36 months old children with either anaemia post-malaria (n = 37) or presumed iron deficiency anaemia alone (n = 36). All children were supplemented for 30 days with 2 mg/kg elemental iron as liquid iron sulphate and administered ⁵⁷Fe and ⁵⁸Fe on days 1 and 15 of supplementation respectively. ⁵⁷Fe and⁵⁸Fe incorporation were significantly reduced (8% vs. 28%: p<0.001 and 14% vs. 26%: p = 0.045) in the malaria vs. non-malaria groups. There was a significantly greater haemoglobin response in the malaria group at both day 15 (p = 0.001) and 30 (p<0.000) with a regression analysis estimated greater change in haemoglobin of 7.2 g/l (s.e. 2.0) and 10.1 g/l (s.e. 2.5) respectively.

Conclusion/Significance

Post-malaria anaemia is associated with a better haemoglobin recovery despite a significant depressant effect on oral iron incorporation which may indicate that early erythropoetic iron need is met by iron recycling rather than oral iron. Supplemental iron administration is of questionable utility within 2 weeks of clinical malaria in children with mild or moderate anaemia.     

A simple and rapid method for the determination of "free" iron in biological fluids

We present a convenient method for determining "free" or non-protein-bound iron in biological fluids. The new method is based on the bathophenantroline method for determination of total serum iron, and comprises binding of iron by a chromogenic chelator (bathophenantroline-disulphonate, BPS), which is specific for ferrous iron. The ferrous complex of BPS absorbs strongly at 535 nm, and the detection limit is less than 1 μM in a sample size of 50 μl. The chelator does not liberate iron from either haemoglobin or transferrin. Interference from copper or zinc in concentrations up to 50 μM does not significantly disturb measurements. The main problem when measuring in blood plasma, the high and fluctuating background in the region around 535 nm, has been overcome through filtering techniques. Data from measurements of ferrous iron in microdialysate, cerebrospinal fluid, and blood plasma in different animal models and clinical conditions are presented as illustrative examples of the usefulness of the method. The method allows the determination of ferric, as well as ferrous, iron in the same sample.     

Erythropoietin effects on iron metabolism in rat bone marrow cells

This paper describes a study of the incorporation of ^{5 9}Fe from ^{5 9}Fe-labelled rat transferrin into rat bone marrow cells in culture. ^{5 9}Fe was found in both stroma and cytoplasm of marrow cells, and the cytoplasmic ^{5 9}Fe separated by polyacrylamide gel electrophoresis, into ferritin, haemoglobin and a low molecular weight fraction.The incorporation of ^{5 9}Fe into all three cytoplasmic fractions, but not into the stroma, increased progressively with time. Erythropoietin stimulated the increase of ^{5 9}Fe in ferritin within 1 h, the earliest time examined, and more than 3 h later in the stroma and haemoglobin.A proportion of the ⁵⁹Fe incorporated into the stroma and low molecular weight iron fractions during a 1 h incubation with ⁵⁹Fe-labelled transferrin was mobilised into ferritin and haemoglobin during a subsequent 4-h “cold-chase”. Erythropoietin, when present during the “cold-chase”, did not influence these ⁵⁹Fe fluxes. The erythropoietin stimulation of ⁵⁹Fe incorporation into ferritin, one of the earliest erythropoietin effects to be recorded, was therefore considered to be due to an increase of ⁵⁹Fe uptake by the hormone-responsive cells rather than a direct effect on ferritin synthesis.20-h cultures containing erythropoietin when incubated with ⁵⁹Fe-labelled transferrin for 4 h, showed dose-related erythropoietin stimulation of ⁵⁹Fe incorporation into haemoglobin only.In the presence of 10 mM isonicotinic acid hydrazide, ⁵⁹Fe incorporation into haemoglobin was inhibited, as in reticulocytes (Ponka, P. and Neuwirt, J. (1969) Blood 33, 690–707), while that into the stroma, ferritin and low molecular weight iron fractions, was stimulated; there were no reproducible effects of erythropoietin.     

The separation of ferritin and haemosiderin for studies in the metabolism of iron

1. Ferritin and haemosiderin in diluted tissue cannot be quantitatively separated by centrifugation unless the pH is brought below 6. 2. These iron-storage substances can be separated from one another and from haemoglobin by ion-exchange chromatography on CM-cellulose, when good recoveries of total tissue iron are obtained. 3. The ferritin fraction has been identified by its solubility, its appearance under the electron microscope, crystallization with Cd²⁺ and the fact that it corresponds quantitatively to the heat-stable portion of the tissue non-haem iron.     

Investigation of the electronic term scheme of deoxygenated human haemoglobin by a least squares fit procedure using simultaneously magnetic susceptibility and Mössbauer data

Summary The calculation of the magnetic susceptibility from a published term scheme for the ferrous iron in deoxygenated human haemoglobin is discussed and a procedure for the simultaneous least squares fit of susceptibility and Mössbauer data is presented. The application of this procedure to the appropriate measurements on human haemoglobin leads to a rearrangement of the low lying electronic levels of the iron. The term schemes received as results of two different sets of susceptibility data used in combination with one set of Mössbauer data overlap with their error bars. The obtained level scheme of the Fe is correlated with the distance of the iron atom from the haem plane and the distance Fe-HIS F8, and some biological implications of these correlations are discussed.     

Genetic parameters for haemoglobin levels in pigs and iron content in pork

Genetic parameters were obtained for iron content in m. longissimus dorsi (2255 records) and haemoglobin levels recorded at 5 (4974 records) and 21 (2405 records) weeks of age in two sire lines from September 2009 until January 2011. The measure of iron in pork was the mean of two replicates. Genetic associations of haematological traits with meat quality traits (2255 records), as well as growth rate and backfat (close to 60 000 records), were estimated. Analyses were based on an animal model using residual maximum likelihood procedures. Iron content in pork was moderately heritable (0.34 ± 0.07) and genetic correlations with haemoglobin measures ranged from 0.39 ± 0.24 to 0.58 ± 0.13, indicating their potential use as selection criteria for increasing iron levels in pork. However, heritabilities for haemoglobin levels were low, ranging from 0.04 ± 0.2 to 0.18 ± 0.04. Procedures to measure haemoglobin on farm may require refinement. Redness of pork, quantified by a* value, had high genetic correlations with iron content (0.90 ± 0.04 to 0.94 ± 0.03) and moderate genetic correlations with haemoglobin levels (0.31 ± 0.22 to 0.55 ± 0.15). Iron content had significant genetic associations with L* measures (−0.61 ± 0.14 to −0.54 ± 0.23), b* value (0.60 ± 0.14 for dorsal b* measure, 0.50 ± 0.15 for average of dorsal and ventral b* measures) and pH at 45 min post mortem (−0.42 ± 0.14). These high genetic correlations between colour measurements and iron content in pork provide further avenues for selection strategies to improve iron content in pork. Current selection practices are not expected to affect iron content in pork, as no significant genetic correlations between performance and haematological traits were found.     

Transport of iron by mycobactin in

The uptake of ⁵⁵FeCl₃ and ⁵⁵Fe-mycobactin has been followed. FeCl₃ was the more effective source of iron for the cell although Fe-mycobactin was adsorbed on to the cell very quickly. The mycobactin within the cell is the prime receptor for Fe³⁺ and it probably functions as an ionophore for the transport of iron across the lipid envelope of the cell. Release of iron from mycobactin occurs by a NADH-dependent reductase forming Fe²⁺ and conditions for observing the activity of this unique enzyme are given.     

Release of iron from haemoglobin--a possible source of free radicals in diabetes mellitus

Increased blood glucose in diabetes mellitus stimulates nonenzymatic glycosylation of several proteins, including haemoglobin. Although iron is tightly bound to haemoglobin, it is liberated under specific circumstances yielding free reactive iron. Studies with purified haemoglobin from normal individuals and diabetic patients revealed that concentration of free iron was significantly higher in the latter cases and increased progressively with extent of the disease. In vitro glycosylation of haemoglobin also led to increase in release of iron from protein. This increase in free iron, acting as a Fenton reagent, might produce free radicals, which, in turn might be causing oxidative stress in diabetes.     

Structure of human deoxy cobalt haemoglobin

The structure of human adult deoxy cobalt haemoglobin has been compared to that of the native ferrous form by refinements based on X-ray data to 2.5 Å resolution. The two structures were refined in parallel by conventional methods and selected structural differences were measured by a novel difference refinement method applicable to closely related structures. The distance between the metal and the haem plane is 0.33 ± 0.08 Å in the cobalt derivative and 0.56 ± 0.03 Å in the native. The Co²⁺HisF8N_? bond length is about 0.1 to 0.2 Å longer than the Fe²⁺HisF8N_? bond length; the distance of N_? from the mean haem plane remains the same in the two structures and the substitution of cobalt for iron produces no significant change in globin structure. The free energy of co-operativity of cobalt haemoglobin is known to be about one-half of that of the native haemoglobin; the reason for this reduction is not evident from the structure of cobalt deoxyhaemoglobin.     

Acquisition of iron from host sources by mesophilic Aeromonas species.

The mesophilic Aeromonas species are opportunistic pathogens that produce either of the siderophores amonabactin or enterobactin. Acquisition of iron for growth from Fe-transferrin in serum was dependent on the siderophore amonabactin; 50 of 54 amonabactin-producing isolates grew in heat-inactivated serum, whereas none of 30 enterobactin-producing strains were able to grow. Most isolates (regardless of siderophore produced) used haem as a sole source of iron for growth; all of 33 isolates grew with either haematin or haemoglobin and 30 of these used haemoglobin when complexed to human haptoglobin. Mutants unable to synthesize a siderophore used iron from haem, suggesting that this capacity was unrelated to siderophore production. Some members of the mesophilic Aeromonas species have evolved both siderophore-dependent and -independent mechanisms for acquisition of iron from a host.     

NO ASSOCIATION BETWEEN tHbmass AND POLYMORPHISMS IN THE HBB GENE IN ENDURANCE ATHLETES

The aim of this study was to examine the association between tHbmass and HBB gene polymorphisms in athletes of endurance disciplines. Eighty-two well-trained athletes (female n=36, male n=46), aged 19.3 ± 2.7 years, representing cross country skiing (n=37) and middle- and long-distance running (n=45), participated in the study. Genotyping for 2 polymorphisms in the HBB gene (- 551C/T and intron 2, +16 C/G) was performed using restriction fragment length polymorphism analysis. Total haemoglobin mass (tHbmass) was determined by the optimized carbon monoxide rebreathing method. Blood morphology, indices of iron status (ferritin, transferrin receptor and total iron binding capacity) and C reactive protein were also determined. No differences were found in the HBB genotype and allele frequencies between male and female athletes. Regardless of the polymorphisms, no relationships were found between HBB genotypes as well as alleles and relative values of tHbmass, expressed per body mass (g · kg^-1 BM), both in female and male athletes. Our results demonstrated that -551 C/T and intron 2, +16 C/G polymorphisms of the HBB gene have no association with total haemoglobin mass in endurance athletes. It cannot be ruled out that several polymorphisms, each with a small but significant contribution, may be responsible for the amount of haemoglobin.     

Affinity chromatography of haemoproteins: 1. Synthesis of various imidazole containing matrices and their interaction with haemoglobin

The functional centre of haemoproteins is generally formed by an iron porphyrin and amino acid residues of he protein component. Some haemoproteins are able to bind imidazole to the iron of the prosthetic group. The synthesis of imidazole containing matrices is described and the affinity of haemoglobin as a model compound to these matrices has been studied. It was found that the lenght and structure of spacers as well as substituents at the imidazole ring are of critical importance: the adsorption of methaemoglobin shows two different kinds of protein matrix interaction: in case of a space length < 5 Å no interaction occurs for steric reasons; at a length > 5 Å adsorption takes place via complex formation between imidazole and iron of a prosthetic group independent of the linkage in 1- or 4(5)-position of the imidazole ring to the mtrix: the complex formation between imidazole and iron is the decisive step but is not solely responsible for the stability of the ocmplex: a hydroxyl group at the side chain near the imidazole decreases the adsorption drastically: large substituents at the imidazole ring disturb complex formation with the iron, but not the adsorption of haemoglobin; in the presence of a long spacer (> 20 A), hydrophobic interactions are predominantly responsible for the adsorption process and imidazole does not play any role.     

The frpB1 gene of Helicobacter pylori is regulated by iron and encodes a membrane protein capable of binding haem and haemoglobin

FrpB1 is a novel membrane protein of Helicobacter pylori that is capable of binding both haem and haemoglobin but consistently shows more affinity for haem. The mRNA levels of frpB1 were repressed by iron and lightly modulated by haem or haemoglobin. The overexpression of the frpB1 gene supported cellular growth when haem or haemoglobin were supplied as the only iron source. Three-dimensional modelling revealed the presence of motifs necessary to bind either haem or haemoglobin. Our overall results support the idea that FrpB1 is a membrane protein of H. pylori that allows this pathogen to survive in the human stomach.     

Non-equilibrium state of a monomeric insect haemoglobin induced by γ-irradiation and detected by Mössbauer spectroscopy

The met-aquo form of the monomeric insect haemoglobin CTT III has been investigated by Mössbauer spectroscopy before and after reduction with thermolyzed electrons at low temperature. The native met haemoglobin dissolved in water and water/glycerol mixtures, respectively, exhibits in the range of pH 5.8 to 9.0 high-spin iron(III). The electronic state of the haemoglobin is not affected by the solvent conditions. In water/glycerol γ-irradiation at 77 K results in the reduction of the haem iron by thermolyzed electrons. Due to this process, the hexacoordinated high-spin iron(III) is transformed into a hexacoordinated low-spin iron(II). This latter complex is a transition state which changes into the high-spin iron(II) state of the deoxyhaemoglobin when increasing the temperature. Thus, a kinetically stabilized non-equilibrium state of the haemoglobin exists at low temperature which relaxes with increasing temperature and finally reaches the equilibrium state to form deoxyhaemoglobin. This transition occurs at T > 190 K and corresponds with drastic changes in the temperature dependence of the Lamb-Mössbauer factor. Both effects indicate an alteration of the intramolecular flexibility of the haemoglobin.     

An endocytic mechanism for haemoglobin-iron acquisition in Candida albicans

The fungal pathogen Candida albicans is able to utilize haemin and haemoglobin as iron sources. Haem-iron utilization is facilitated by Rbt5, an extracellular, glycosylphophatidylinositol (GPI)-anchored, haemin- and haemoglobin-binding protein. Here, we show that Rbt5 and its close homologue Rbt51 are short-lived plasma membrane proteins, degradation of which depends on vacuolar activity. Rbt5 facilitates the rapid endocytosis of haemoglobin into the C.  albicans vacuole. We relied on recapitulation of the Rbt51-dependent haem-iron utilization in Saccharomyces cerevisiae to identify mutants defective in haemoglobin utilization. Homologues of representative mutants in S. cerevisiae were deleted in C. albicans and tested for haemoglobin-iron utilization and haemoglobin uptake. These mutants define a novel endocytosis-mediated haemoglobin utilization mechanism that depends on acidification of the lumen of the late secretory pathway, on a type I myosin and on the activity of the ESCRT pathway.     

Transferrin-bipyridine iron transfer mediated by haemoproteins

Rabbit reticulocyte cytosol was able to mediate transferrin-bipyridine iron transfer in the presence of ATP. The cytoplasmic factor responsible for the mediation of iron transfer was identified as haemoglobin. Other cytoplasmic proteins and the membrane fraction were ineffective. Human α and β subunits and human myoglobin were over three times more effective than human haemoglobin A. Carbon monoxide strongly inhibited the mediation of iron transfer. Oxidation of haemoglobin abolished it but methaemoglobin could be reactivated with NADH, even when azide was bound to the haem iron.Neither globin nor haem alone were able to mediate iron transfer, even when NADH was present. Together, the reconstituted methaemoglobin A could be reactivated with NADH.Although the physiological significance of this phenomenon is not clear, the involvement of haemoproteins in intracellular iron metabolism seems likely.     

Increased Gastric IL-1β Concentration and Iron Deficiency Parameters in H. pylori Infected Children

Association between H. pylori infection, iron deficiency and iron deficiency anaemia has been described, but the mechanisms involved have not been established. We hypothesized that in H. pylori infected children increased gastric concentrations of IL-1β and/or TNF-α, both potent inhibitors of gastric acid secretion that is essential for iron absorption, are predictors for low blood concentrations of ferritin and haemoglobin, markers of early depletion of iron stores and anaemia, respectively. We evaluated 125 children undergoing endoscopy to clarify the origin of gastrointestinal symptoms. Gastric specimens were obtained for H. pylori status and cytokine evaluation and blood samples for determination of iron deficiency/iron deficiency anaemia parameters and IL1 cluster and TNFA polymorphisms that are associated with increased cytokine secretions. Higher IL-1β and TNF-α gastric concentrations were observed in H. pylori-positive (n = 47) than in -negative (n = 78) children. Multiple linear regression models revealed gastric IL-1β, but not TNF-α, as a significant predictor of low ferritin and haemoglobin concentrations; results were reproduced in young children in whom IL1RN polymorphic genotypes associated with higher gastric IL-1β expression and lower blood ferritin and haemoglobin concentrations. In conclusion, high gastric levels of IL-1β can be the link between H. pylori infection and iron deficiency/iron deficiency anaemia in childhood.