Iron absorption in the iron-deficient rat.

Iron absorption in the iron-deficient rat was compared with that in the normal rat to better understand the regulation of this dynamic process. It was found that: Iron uptake by the iron-deficient intestinal mucosa was prolonged as a result of slower gastric release, particularly when larger doses of iron were employed. The increased mucosal uptake of ionized iron was not the result of increased adsorption, but instead appeared related to a metabolically active uptake process, whereas the increased mucosal uptake of transferrin iron was associated with increased numbers of mucosal cell membrane transferrin receptors. Mucosal ferritin acted as an iron storage protein, but its iron uptake did not explain the lower iron absorption in the normal rat. Iron loading the mucosal cell (by presenting a large iron dose to the intestinal lumen) decreased absorption for 3 to 4 days. Iron loading of the mucosal cell from circulating plasma transferrin was proportionate to the plasma iron concentration. Mucosal iron content was the composite of iron loading from the lumen and loading from plasma transferrin versus release of iron into the body. These studies imply that an enhanced uptake-throughout mechanism causes the increased iron absorption in the iron-deficient rat. Results were consistent with the existence of a regulating mechanism for iron absorption that responds to change in mucosal cell iron, which is best reflected by mucosal ferritin.

Iron is not required in the lactoferrin stimulation of thymidine incorporation into the DNA of rat crypt enterocytes.

Lactoferrin has been identified as a factor in human colostrum that accounts for increased incorporation of thymidine into the DNA in an in vitro rat crypt enterocyte bioassay. We have examined lactoferrin-stimulated thymidine incorporation by comparing the effects of iron-free lactoferrin (apolactoferrin) with those of iron-saturated lactoferrin (diferric lactoferrin) under conditions that inhibit the transfer of iron between these iron-binding proteins in the bioassay system. In addition, we have compared the dose-response relationships of diferric lactoferrin and apolactoferrin. The results demonstrated that lactoferrin, independent of iron-binding states, promoted the incorporation of thymidine into the DNA of rat crypt enterocytes. These observations suggest a previously unreported nutritional role for lactoferrin that is independent of its iron-binding capacity.

Transferrin-reticulocyte cycle time in rat reticulocytes.

The uptake and release of 131I-labelled diferric transferrin by rat reticulocytes was examined both in vitro and in vivo. Cycle time in vitro was estimated to be 2.5 min in iron-deficient reticulocytes and 2.3 min in phenylhydrazine-produced reticulocytes. In vivo reticulocyte uptake and release of labelled diferric transferrin injected in the iron-deficient rat averaged 1.7 min.

Intact transferrin receptors in human plasma and their relation to erythropoiesis.

Intact transferrin receptor molecules complexed with transferrin were found in human plasma. The concentration of receptors was determined by an enzyme-linked immunosorbent assay that uses polyclonal antibodies. The mean concentration of 8,279 micrograms/L in 56 normal adults appears to be unrelated to age or sex. Additional receptor measurements were performed on plasmas from 260 subjects with erythropoietic disorders. Decreased concentration of plasma receptors was found in patients with erythroid hypoplasia and increased numbers in those with erythroid hyperplasia. Ferrokinetic measurements of erythropoiesis were compared with numbers of receptors in 148 subjects, and a close correlation was found (r = .86). Both sets of values, measured in different conditions and expressed in relation to normal, were consistent with expected values. Receptor values were unproportionally increased only in conditions of iron deficiency. It is concluded that plasma receptors have a constant relationship to tissue receptors, and their number in most instances reflects the rate of erythropoiesis.

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.

Hepatocyte iron release in rats.

Hepatocyte iron release was studied in vivo in rats. After the injection of iron 59-labeled ferritin, hemoglobin, or human asialotransferrin, the proportions of the radioactive iron returned to the plasma and incorporated into stores were determined under various conditions. Iron 55-labeled rat transferrin was injected at the same time as the 59Fe-labeled compound, and storage iron release was calculated from the cumulative incorporation of the two isotopes in the red cell mass over 2 weeks. The various 59Fe-labeled compounds were processed differently by the hepatocyte, but the radioactive iron was incorporated in the same iron stores. About 6% of the hepatocyte storage iron was released daily in normal rats, but a pool of iron that is not mobilized spontaneously was clearly identified in iron overload. Iron turnover in the hepatocyte was regulated by the rate of erythropoiesis and iron status of the animal, and inflammation blocked hepatocyte iron release. A strong correlation between hepatocyte iron release and plasma transferrin receptor levels was observed (p less than 0.001), suggesting that plasma transferrin receptors could mediate the regulation of hepatocyte iron mobilization in rats.

The behavior of asialotransferrin-iron in the rat.

The effect of desialylation of rat and human transferrins on hepatocyte processing of the protein and its iron was studied in rats. No alteration in early transferrin catabolism was observed. Radioiron disappearance from the plasma and liver iron uptake were more rapid for asialotransferrins than for normal transferrins (P less than .001). Furthermore, radioiron plasma clearance of human tri-sialotransferrin was faster (P less than .05) and liver uptake higher (P less than .002) than for human pentasialotransferrin. When the asialoglycoprotein receptor was blocked by the prior injection of asialofetuin, asialotransferrin behaved like normal transferrin. When the transferrin receptor was blocked by the prior injection of 50 mg human diferric transferrin, iron uptake from all transferrins was delayed to such an extent that uptake through both receptors seemed to be affected. Approximately 90% of the hepatic radioiron from all transferrins was chelated by desferrioxamine and excreted into the bile, indicating its uptake by the hepatocyte rather than the reticuloendothelial (RE) cell. The rate of iron release into the plasma and its subsequent accumulation in the red cell mass over a 2-week period was similar for human asialotransferrin, ferritin, and hemoglobin iron. This study 1) confirmed that asialotransferrin-iron uptake by the hepatocyte is mediated by both transferrin and asialoglycoprotein receptors; 2) demonstrated that not only asialotransferrin but also transferrin of low sialic acid content will increase iron turnover and lead to excessive iron loading of the hepatocyte; 3) and showed that the intrahepatocyte metabolism of asialotransferrin-iron did not differ from that of iron delivered by normal transferrin.

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.

In vivo transferrin-iron receptor relationships in erythron of rats.

Quantitative measurements of transferrin receptors, tissue transferrin, tissue iron uptake, and erythroid cellularity have been carried out in rats with altered erythropoiesis and altered iron balance. Erythroid receptors increased with erythroid hyperplasia, with the increase in proportion to the increased number of red cell precursors in phenylhydrazine-treated rats. Receptors increased disproportionately in iron deficiency due to both erythroid hyperplasia and an increase in receptors in the individual cell. There was a ratio of 1:1 between cell-related transferrin and receptors in circulating reticulocytes but a disproportionate amount of cell-related transferrin in fixed erythroid tissues (marrow and spleen), suggesting that there was some other reason for the concentration of transferrin in these tissues. Erythron iron uptake was increased in proportion to the increased receptor number in phenylhydrazine-treated animals but was reduced in iron deficiency because of the limited amount of iron-bearing transferrin. These studies demonstrate the dominant role of erythron cellularity and iron status in vivo in determining total receptor number and the importance of receptor number and iron supply in tissue iron uptake.

Quantitation of ferritin iron in plasma, an explanation for non-transferrin iron.

In 33 patients with thalassemia and idiopathic hemochromatosis, plasma ferritin protein levels ranged from 36 to 5,850 micrograms/L. The iron content of this ferritin as determined by immunoprecipitation ranged from undetectable amounts to 507 micrograms/L. The mean iron content of ferritin protein in those and other subjects with plasma ferritin concentrations of over 1,000 was 6.8% +/- 2.7%. Plasma transferrin was usually saturated with iron in patients with measurable ferritin iron, but exceptions occurred. In studies using electrophoretic separation, it was shown that some ferritin iron moved to transferrin during in vitro incubation, whereas exchange in the opposite direction was extremely limited. Because some plasma ferritin iron was measured by the standard colorimetric plasma iron determination, these observations (a) indicate that plasma ferritin contains a significant amount of iron (b) indicate that a significant proportion of nontransferrin iron in individuals with nontransferrin iron as detected by standard plasma iron and total iron-binding capacity measurements is due to the presence of ferritin, and (c) suggest that large amounts of ferritin iron may affect the saturation of plasma transferrin.

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

Manduca sexta larvae accumulate large amounts of iron during their larval feeding period. When 59Fe 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 iron-containing 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 micrograms 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 in Manduca sexta, the latter serving a storage function during development and possibly also a transport function.

Transferrin receptors in rat plasma.

Antigenic material in rat plasma reacting with rat transferrin receptor antibodies was identified as an intact receptor molecule complexed with transferrin. Plasma transferrin receptors were measured by ELISA in rats of different age and sex, of different iron status, with different degrees of erythropoiesis, and with inflammation. An inverse relationship between iron status and receptor number was found, whereas a direct relationship existed between erythropoiesis and receptors. These changes in receptor number can be explained by assuming that the number of tissue receptors determined the number of plasma receptors and that the erythroid cells possessed most of the body's receptors. Increases in plasma receptors lagged behind the appearance of circulating reticulocytes, suggesting that receptors were released to the plasma during the terminal phase of erythrocyte maturation.

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.

Removal of transferrin from fetal bovine serum.

An antiserum against purified fetal bovine serum (FBS) transferrin was produced in rabbits. The isolation of anti-bovine transferrin IgG fraction was achieved by ammonium sulfate precipitation of rabbit hyperimmune plasma followed by ion exchange chromatography on diethylaminoethanol (DEAE) cellulose, at both an acidic and basic pH. The various antibody fractions were analyzed by fast protein liquid chromatography (FPLC) on a high-resolution mono-Q column. The specificity and efficiency of the antibody fractions obtained were tested by titration of a constant amount of fetal bovine serum with increasing amounts of antibody. The completeness of removal of the fetal bovine serum transferrin resulting from the formation of the highly stable antibody antigen complex was monitored by immunologic and radioisotope methods. The removal of FBS transferrin by this antibody precipitation technique did not interfere with the ability of added iron 59-tagged human transferrin to deliver iron to rat reticulocytes, as shown in an in vivo incubation model. The method proved to be effective for the fast and complete removal of bovine transferrin from fetal bovine serum in vitro and will be a prerequisite for more detailed studies of the interaction of transferrin of different species with tissue receptors on cell lines in culture without resorting to completely defined culture media.

Adequacy of iron supply for erythropoiesis: in vivo observations in humans.

The adequacy of tissue iron supply was examined with ferrokinetic techniques in subjects with decreased plasma iron concentration and in subjects with a normal plasma iron concentration but with increased tissue iron requirements. The competition by transferrin receptors for diferric vs monoferric transferrin was measured in eight normal persons and eight with iron deficiency. There was a highly significant (P less than 0.001) decrease in receptor preference for diferric transferrin in subjects with iron deficiency, indicating an insufficient amount of iron-bearing transferrin to saturate tissue receptors. The adequacy of the plasma iron supply was also examined by determining the number of iron-bearing transferrin molecules with receptors at normal and elevated plasma iron concentrations. Significant increases were found at the higher plasma iron concentration, not only in patients with iron deficiency, but also in patients with sickle cell anemia and thalassemia. Furthermore, the increase in the latter two groups was shown to be proportional to the degree of erythroid hyperplasia. These data indicate that tissue iron supply must be evaluated in terms of both plasma iron supply and erythropoietic requirements and that a relative iron deficiency is frequent in patients with erythroid hyperplasia.

Interpreting results of coulometry and immunoprecipitation in diagnosing iron disorders.

Concentration of iron in plasma, total iron-binding capacity (TIBC), and transferrin saturation are often determined by standard spectrophotometric methods, but iron concentration may be quantified by immunoprecipitation or, electrochemically, by controlled-potential coulometry. Because these iron assays do not all measure the same form(s) of iron, we studied subjects in various states of iron nutriture: normal adults, iron-deficient patients, thalassemia patients with unsaturated transferrin or oversaturated transferrin, and patients with idiopathic hemochromatosis. The spectrophotometric and coulometric methods detected essentially all non-heme iron in plasma; results correlated well but showed a negative bias toward the coulometric method. Results by an immunoprecipitation procedure, which measures only transferrin-bound iron, correlated well with those obtained coulometrically but were slightly higher than the latter. The characteristics of the various methods for iron must be understood by the clinical laboratory if diagnosis of iron disorders is to be accurate.

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.

The cadmium effect on iron absorption.

Test solutions of cadmium and labeled iron salts, soluble complexes of diferric transferrin, or hemoglobin iron were introduced orally or were injected into tied-off jejunal segments in rat. Cadmium reduced the absorption of iron salts to about half in both normal and iron-deficient rats. Hemoglobin iron absorption was enhanced, indicating that the processing of this form or iron and its release from mucosa to blood was intact. A greater reduction in iron absorption occurred in iron-deficient rats when transferrin iron was injected into gut loops. Mucosal radioiron content in animals given cadmium with either iron salts or transferrin iron was increased. The primary effect of cadmium was on intracellular processing of iron salts and transferrin iron. The major portion of cadmium taken up by the mucosa of normal animals was bound to ferritin, and the effect of cadmium within the mucosal cell may be reduced thus.