Helmut A. Huebers

Iron binding proteins and their roles in the tobacco hornworm, Manduca sexta (L.).

SummaryManduca sexta larvae accumulate large amounts of iron during their larval feeding period. When59Fe was fed to 5th instar larvae, it was evenly distributed among the hemolymph, gut and carcass until the cessation of feeding. By pupation 95% of the labelled iron was found in the fat body. In the adult a significant portion of this iron was found in flight muscle.Studies of the hemolymph disclosed two ironcontaining proteins. The first was composed of a single polypeptide chain of 80 kD, containing one atom of iron. This protein bound ionic iron in vitro and was able to transfer this iron to ferritin when incubated with fat body in vitro. Therefore, it appeared to serve a transport function. The second protein had a molecular weight of 490 kD with subunits of 24 and 26 kD and contained 220 μg of iron/mg protein. Its chemical and ultrastructural characteristics were those of ferritin. These studies demonstrate the presence of both a transport protein and a unique circulating ferritin inManduca sexta, the latter serving a storage function during development and possibly also a transport function.

Characterization of an invertebrate transferrin from the crabCancer magister (Arthropoda)

SummaryAn iron binding protein was isolated from crab blood and tested for its chemical and functional behavior.1.The protein has a molecular weight of 150,000±10,000 and consists of a single polypeptide chain. The iron content was found to be two molecules iron per molecule protein; the presence of a 52-fold molar excess of copper did not alter the iron binding properties.2.Spectroscopical analysis carried out in the presence of bicarbonate yielded two absorption maxima at λ=276 and λ=465 nm.3.The59Fe-tagged crab protein did not yield its iron to human apotransferrin when incubated in human plasma. When incubated with rat reticulocytes, the crab iron protein delivered its iron for hemoglobin and ferritin synthesis. This delivery can be completely abolished by adding an excess of rat transferrin.4.When injected into rats in vivo, the crab protein delivered its iron to erythroid and non-erythroid tissue. The amount delivered to the erythroid marrow was shown to depend on iron requirements of that tissue.5.When injected as59Fe-tagged protein into a crab, half-time disappearance of the radioiron is reached between 7 and 9 h. In the liver and in the carapace lining, the radioiron released is found in other iron binders.6.Except for the significantly larger molecular weight, the crab iron binding protein fulfills all the criteria for transferrin. The findings negate the concept that transferrin is a newcomer in the evolutionary scene and to be found only in the phylum Chordata. Thus the need for a specific iron transport protein of the transferrin class is independent of the achievement of hemoglobin as a means to accomplish oxygen transport.

Molecular advantage of diferric transferrin in delivering iron to reticulocytes: a comparative study

Abstract The delivery of transferrin iron from four animal species and man to homologous reticulocytes was measured at different transferrin saturations. Total iron uptake in the in vitro reticulocyte incubation model employed followed a hyperbolic curve, increasing as the transferrin saturation increased but at a progressively slower rate. In all species, there was a much greater iron delivery from diferric as compared to monoferric transferrin, the molecular advantage varying from 8:1 to 14:1. The majority of iron was delivered from diferric transferrin when transferrin saturations exceeded 13-19% depending on the species. Thus a general similarity exists in the transferrin-iron receptor interactions in these mammalian species. Formuli have been provided whereby the iron utilization curve may be calculated when uptake has been determined at any one transferrin saturation.

Ferrokinetic Measurement of Erythropoiesis

Ferrokinetic measurements have proved useful because of the dominant role of the erythron in tissue iron uptake. Detailed measurements of the plasma iron disappearance curve coupled with in vivo counting have defined the major pathways of iron utilization and early refluxes of iron into plasma. Recent studies have disclosed two separate plasma kinetic pools consisting of mono- and diferric transferrin, and have demonstrated the effect of their relative abundance on tissue iron uptake. Allowance for the amount of each has made possible the calculation of iron-bearing transferrin uptake, which is independent of plasma iron concentration as long as receptors are saturated. This refinement permits the measurement of functional erythron transferrin receptors, and thereby the relative number of immature erythroid cells.

Iron metabolism in a spider,Dugesiella hentzi

SummaryBody iron content and iron balance were studied in the spider (D. hentzi). 1.Total iron content of individual male and female spiders averaged 350 μg.2.Food intake consisted of domestic crickets. Iron content of crickets averaged 12.4 μg/cricket of which 8.7 μg was absorbed by the spiders. The average number of crickets ingested during a period of six months was 14, amounting to 124 μg of iron absorbed.3.Most of the iron was localized in the opisthosoma. In four female spiders a mean of 85% of the radioiron still present 13 to 15 months after ingestion was found there.4.Iron excretion in urine and feces was 19.9 μg/month or 119 μg for a six month period. The excretion of radioiron followed in four females showed a halflife of six months.5.Molts showed an average iron content of 41.3 μg. These large spiders may molt every year or at considerably longer intervals. If iron lost in the molt is added to other iron loss, it appears to exceed the estimated intake, but by a negligible fraction.6.Cell free normal blood revealed that iron is present to the extent of 85 μg/100 ml and is bound to two proteins. Some of the properties of the proteins are reported. Saturation with radioiron showed that the blood could take up another 21 μg iron/100 ml.

The Effect of Transferrin Saturation on Internal Iron Exchange

Abstract Radioiron was introduced into the intestinal lumen to evaluate absorption, injected as nonviable red cells to evaluate reticuloendothelial (RE) processing of iron, and injected as hemoglobin to evaluate hepatocyte iron processing. Redistribution of iron through the plasma was evaluated in control animals and animals whose transferrin was saturated by iron infusion. Radioiron introduced into the lumen of the gut as ferrous sulfate and as transferrin-bound iron was absorbed about half as well in iron-infused animals, and absorbed iron was localized in the liver. The similar absorption of transferrin-bound iron suggested that absorption of ferrous iron occurred via the mucosal cell and did not enter by diffusion. The decrease in absorption was associated with an increase in mucosal iron and ferritin content produced by the iron infusion. An inverse relationship (r = —0.895) was shown between mucosal ferritin iron and absorption. When iron was injected as nonviable red cells, it was deposited predominantly in reticuloendothelial cells of the spleen. Return of this radioiron to the plasma was only 6% of that in control animals. While there was some movement of iron from spleen to liver, this could be accounted for by intravascular hemolysis. Injected hemoglobin tagged with radioiron was for the most part taken up and held by the liver. Some 13% initially localized in the marrow in iron-infused animals was shown to be storage iron unavailable for hemoglobin synthesis. These studies demonstrate the hepatic trapping of absorbed iron and the inability of either RE cell or hepatocyte to release iron in the transferrin-saturated animal.

A highly efficient chemical isolation procedure for the rat placental transferrin receptor.

A chemical method for the purification of rat placental transferrin receptor is described. After initial solubilization and concentration by ammonium sulfate precipitation, radioiron-tagged diferric transferrin was added to the dialyzed receptor fraction and subjected to anion-exchange chromatography on DEAE-Sephacel. Elution with a Tris-HCl buffer gradient yields a single fraction of radioactivity containing both free transferrin and the receptor-transferrin as a complex. Further separation of the receptor-transferrin complex from the free transferrin is achieved by gel chromatography on a AcA34-Sepharose 6B separation system. Final purification is obtained by preparative gel electrophoresis in 5% polyacrylamide gels. The receptor was shown to be pure by various methods including HPLC chromatography. The average yield was 20-30 mg receptor-transferrin complex/100 g placental tissue. Because of the purely chemical approach, this method is universally applicable for the isolation of transferrin receptors from various tissues.

Blocking action of parenteral desferrioxamine on iron absorption in rodents and men

Desferrioxamine (DFO) is an iron chelating agent that, when administered orally, interferes with gut absorption of inorganic iron and, when administered parenterally, binds body iron and is excreted as ferrioxamine in bile and urine. Studies were carried out in normal and iron-deficient male rats and in normal, iron-replete male volunteers to investigate the blocking action of parenteral DFO on the absorption of radioiron. Radiolabeled ferrous ammonium sulfate, transferrin iron, or hemoglobin iron was injected directly into the jejunum of rats with or without intramuscular injections of DFO. Radioiron administered as ferrous sulfate or as transferrin iron was given to the volunteers by mouth or by direct duodenal infusion, respectively, with or without intravenous infusions of DFO. In iron-deficient rats, intramuscular DFO injections commencing 1 h before direct jejunal injection of radioiron significantly blocked absorption of inorganic iron (26% with DFO, 64% without DFO), transferrin iron (4% with DFO, 69% without DFO), and hemoglobin iron (3% with DFO, 19% without DFO). In normal rats, DFO injections also significantly blocked absorption of inorganic iron and transferrin iron. In normal volunteers, intravenous DFO infusions commencing 1 h before administration of radioiron significantly blocked absorption of physiologic doses of inorganic iron (3% with DFO, 21% without DFO) and transferrin iron (1% with DFO, 20% without DFO). The quantity of radioiron excreted in urine by both rats and humans with administration of DFO did not account for the observed decrement in absorption of radioiron. Biochemical analysis of rat intestinal mucosal scrapings after injection of DFO and administration of radioiron demonstrated the accumulation of a small molecular weight fraction containing iron that was ferrioxamine (iron-chelate) complex. We conclude that parenterally administered DFO can enter the small intestinal mucosa, bind intracellular iron, and block iron absorption. Parenteral DFO blocks the absorption of inorganic iron, transferrin iron, and hemoglobin iron, suggesting that all three iron species enter a common chelatable pool within the small intestinal mucosa and may share a common pathway of absorption.

Random distribution of iron among the two binding sites of transferrin in patients with various hematologic disorders

The distribution of iron among the two binding sites of transferrin was studied by isoelectric focusing in 41 patients with a variety of disorders of iron metabolism and erythropoiesis. The proportion of diferric and the two monoferric transferrins were very close to the values predicted from the transferring saturation. It is concluded that iron distribution on transferrin is random or close to random within the experimental error in patients with a variety of clinical disorders.

Pathophysiological classification of acquired bone marrow failure based on quantitative assessment of erythroid function

Bone marrow failure encompasses a broad spectrum of disorders including aplastic, dysmyelopoietic and myelophtisic anemias. In the present study, these anemias were characterized according to the degree of erythroid proliferation and efficiency of erythropoiesis. Total erythropoietic activity was evaluated in 43 patients by measuring the erythron transferrin uptake (ETU). It averaged 20% of basal (range 3–43%) in 13 patients with severe aplastic anemia, 75% of basal (range 60–103%) in 3 patients with extensive bone marrow infiltration by neoplastic cells, 131 % of basal (range 50–217%) in 16 patients with refractory anemia, and 452% of basal (range 63–720) in 11 patients with idiopathic refractory siderobastic anemia. Respective efficiencies of erythropoiesis were 74% in aplastic anemia, 70% with bone marrow infiltration, 46% in refractory anemia, and 14% in sideroblastic anemia. Based on the ETU, patients could be categorized into absolute marrow failure, relative marrow failure, and adequate erythropoietic response to anemia. This simple determination of proliferating activity of the erythroid marrow can provide useful information on the pathophysiology of marrow failure and a basis for the selection of therapeutic approaches.