Hepatic Iron Deposition in Humans: I. First-Pass Hepatic Deposition of Intestinally Absorbed Iron in Patients with Low Plasma Latent Iron-Binding Capacity

In patients with spontaneous chronic low plasma latent iron-bindingcapacity, a large fraction of the intestinally absorbed iron is deposited in theliver without prior appearance in the systemic plasma. Such hepatic iron isnot released to any large extent during the subsequent 2 weeks.

Our results are consistent with the hypothesis that iron absorbed from theintestinal tract is not initially bound to transferrin. When the plasma latentiron-binding capacity is normal, iron binds to transferrin prior to reachingthe liver; when the plasma latent iron-binding capacity is low, a portion ofthe iron reaches the liver either unbound or abnormally bound to transferrin,resulting in its immediate hepatic deposition.

Submitted on November 21, 1966 Accepted on March 19, 1967     

Mucosal Iron-Binding Proteins in Mice with X-Linked Anaemia

The mouse with X-linked anaemia (sla) has a defect in iron absorption which can be temporarily reversed by feeding a low iron diet. Duodenal non-haem iron was significantly higher in the sla than in the normal mouse on an iron supplemented diet but non-haem iron was reduced to minute amounts when the mice were fed a low iron diet. Gel chromatography on Sephadex G-200 of the particle-free supernatant of pooled mucosal homogenates revealed the presence of three proteins binding ⁵⁹Fe. Fraction I (mol wt 450 000) resembled ferritin and was present in both normal and sla mice fed an iron supplemented diet. Fraction II (mol wt 78 000) eluted in a similar position to transferrin and was evident in both normal and sla mice fed an iron deficient diet. Fraction III (mol wt less than 15 000) contained equivalent amounts of radioiron in normal and sla mice fed the iron deficient diet, whereas this fraction contained less radioactivity in sla animals in two of three experiments in which the animals were fed an iron supplemented diet. The iron transport defect in sla mice does not appear to reside in the iron-binding proteins in the supernatant fraction of the intestinal mucosa and the cause of the defect in iron absorption remains to be elucidated.     

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.     

Nontransferrin-bound iron in plasma from hemochromatosis patients: effect of phlebotomy therapy

Plasma from patients with iron overload resulting from idiopathic hemochromatosis contains nontransferrin-bound iron, measurable by the bleomycin, assay. During venesection therapy, the concentration of bleomycin iron declines in a way highly correlated with plasma ferritin concentrations. Even when patients had been venesected to give very low total plasma iron concentrations and high transferrin iron-binding capacity, bleomycin-detectable iron was still present at low concentrations. Bleomycin-detectable iron can stimulate damaging free radical reactions, and its persistence in plasma even after prolonged venesection might contribute to the tissue damage that results from iron overload.     

Iron-binding proteins and free iron in synovial fluids of rheumatoid arthritis patients

Summary Iron-binding proteins (lactoferrin, transferrin and ferritin) and free iron were measured in synovial fluid (SF) from 30 patients with rheumatoid arthritis (RA) and 20 osteoarthritis (OA) patients. The iron-binding proteins except transferrin were significantly increased in RA SF as compared with OA SF. Similarly, free iron was also significantly higher in RA SF than in OA SF, whereas the ferritin saturation index, transferrin saturation index and bound iron were more significantly decreased in RA SF than in OA SF. These results suggest that RA SF contains sufficient micromolar amounts of free iron to allow hydroxyl radical formation. Also the capacity of iron-binding proteins to bind free iron is inadequate in the presence of a large amount of iron-binding proteins which are present in RA SF.     

Effect of aerosolized Escherichia coli and Staphylococcus aureus on iron and iron-binding proteins in lung lavage fluid

Iron-binding proteins have antibacterial activity; they have been identified in lung secretions, but their role in pulmonary antibacterial defenses is unclear. Murine lactoferrin and murine transferrin were used to generate polyclonal antiserum to lactoferrin and to transferrin, and the specificity of both antisera was shown by western blot. Mice were exposed to either aerosolized Escherichia coli or Staphylococcus aureus; they were killed 1, 4, 24, or 48 hr later; and their lungs were lavaged. We measured the levels of transferrin, lactoferrin, and albumin and did a cell count for the lavage fluid. The predominant iron-binding protein in resting animals was transferrin. Aerosolized E. coli caused a brisk PMNL response in the lungs that was associated with a major increase in the levels of lactoferrin. Challenge with S. aureus was associated with a moderate increase in the number of macrophages and a moderate decrease in the levels of transferrin and iron but no change in the levels of lactoferrin. The levels of iron-binding protein can vary according to the type of inflammatory response.     

In vitro and in vivo studies of iron delivery by human monoferric transferrins

S ummary . According to the Fletcher-Huehns hypothesis there exists a functional difference between the two iron-binding sites of transferrin. In this study we present the results of an evaluation of this hypothesis in vitro and in vivo with human pure monoferric transferrins obtained by preparative isoelectric focusing in granulated gels. The uptake of iron from monoferric transferrins TfFe_c and Fe_NTf by erythroid bone marrow cells, hepatocytes and stimulated T-lymphocytes in vitro was equal, even when both monoferric transferrins were present together in the incubation medium. Ferrokinetic studies in vivo , performed with both pure monoferric transferrins, showed that transferrin TfFe_c, as well as transferrin Fe_NTf, mainly deliver their iron to the erythron. As red cell ⁵⁹Fe utilization, red cell iron turnover and other ferrokinetic parameters, obtained from this study, were identical too it is evident that both iron-binding sites of transferrin are functionally homogeneous in vivo , with respect to iron delivery.     

Transferrin in a cockroach: molecular cloning, characterization, and suppression by juvenile hormone.

In a study of juvenile hormone-regulated gene expression, we isolated an anonymous cDNA representing a message that was strongly suppressed by juvenile hormone in the fat body of the cockroach Blaberus discoidalis. The protein deduced from the cDNA sequence showed compelling resemblance in sequence to the transferrins, a superfamily of internally duplicated, 80-kDa iron-binding/transport proteins characterized from several vertebrates and, to date, one insect (the tobacco hornworm, Manduca sexta). We isolated a 78-kDa protein from cockroach hemolymph, verified its congruence with the cloned cDNA, and found that it did bind iron. The cockroach protein is a member of the transferrin superfamily based on several features, including 32-46% amino acid positional identity with transferrins whose sequences are known, internal homology, positioning of cysteine residues, and iron binding. Whereas the previously characterized insect transferrin binds one atom of iron per protein molecule, B. discoidalis transferrin binds two iron atoms as do the vertebrate transferrins. The diferric property of cockroach transferrin is consistent with presence of two sets of residues positioned appropriately for iron binding. Juvenile hormone suppressed transferrin mRNA levels drastically in the adult female cockroach.     

Abnormal in vitro function of a variant human transferrin

Normal and variant transferrins have been isolated from the plasma of an individual heterozygous for the abnormal protein. The normal and variant proteins were separated, saturated with 59Fe, labelled with 125I and then compared in their interaction with PHA-stimulated human lymphocytes in vitro. At 4 degrees C the variant protein bound to the transferrin receptors on these cells with an association constant (6.25 +/- 2.53 X 10(7) l mol-1, mean +/- SD; n = 4) which was an order of magnitude lower than that of the normal diferric transferrin (6.31 +/- 1.82 X 10(8) l mol-1, mean +/- SD; n = 4). This low association constant was reflected in a much reduced rate of iron donation to the cells at 37 degrees C (22.2 +/- 8.3 pg/10(6) cells/h compared with 48.1 +/- 15.6 pg/10(6) cells/h). As the variant transferrin has both abnormal iron-binding properties (Evans et al, 1982) and an abnormal interaction with the transferrin receptor, it would appear that these two functions may be closely interdependent.     

Biological variability of transferrin saturation and unsaturated iron-binding capacity

Background

Transferrin saturation is widely considered the preferred screening test for hemochromatosis. Unsaturated iron-binding capacity has similar performance at lower cost. However, the within-person biological variability of both these tests may limit their ability at commonly used cut points to detect HFE C282Y homozygous patients.

Methods

The Hemochromatosis and Iron Overload Screening Study screened 101,168 primary care participants for iron overload using transferrin saturation, unsaturated iron-binding capacity, ferritin, and HFE C282Y and H63D genotyping. Transferrin saturation and unsaturated iron-binding capacity were performed at initial screening and again when selected participants and controls returned for a clinical examination several months later. A missed case was defined as a C282Y homozygote who had transferrin saturation below the cut point (45% for women, 50% for men) or unsaturated iron-binding capacity above the cut point (150 μmol/L for women, 125 μmol/L for men) at the initial screening or the clinical examination, or both, regardless of serum ferritin.

Results

There were 209 C282Y previously undiagnosed homozygotes with transferrin saturation and unsaturated iron-binding capacity testing performed at the initial screening and clinical examination. Sixty-eight C282Y homozygotes (33%) would have been missed at these transferrin saturation cut points (19 men, 49 women; median serum ferritin level of 170 μg/L; first and third quartiles, 50 and 474 μg/L), and 58 homozygotes (28%) would have been missed at the unsaturated iron-binding capacity cut points (20 men, 38 women; median serum ferritin level of 168 μg/L; first and third quartiles, 38 and 454 μg/L). There was no advantage to using fasting samples.

Conclusions

The within-person biological variability of transferrin saturation and unsaturated iron-binding capacity limits their usefulness as an initial screening test for expressing C282Y homozygotes.     

Effect of transfused reticulocytes on iron exchange

A animal model was developed whereby reticulocyte-rich blood was introduced into normal rats by exchange transfusion. Measurements of plasma iron turnover was made at similar plasma iron concentrations before and after exchange transfusions. High reticulocyte blood obtained from animals rendered iron deficient by diet or by treatment with phenylhydrazine resulted in a mean increase of 86% in internal iron exchange, while the plasma iron turnover was unaffected by exchange with normal red cells. Since iron input from reticuloendothelial cells could have increased due to breakdown of transfused cells, iron absorption was also measured. Within 1 hr and for a least 6 hr after exchange with high reticulocyte blood, mean absorption in six groups of animals was increased over control animals by 50%-130%. The increased plasma iron turnover and absorption was not mediated by a decrease in plasma iron or an increase in unsaturated iron-binding capacity. Indeed, a higher plasma iron and transferrin saturation augmented the movement of iron into the plasma from iron- donating tissues. It is proposed that the donation of iron by transferrin in some way immediately facilitates the procurement of more iron by transferrin.     

The Effect of pH upon Human Transferrin: Selective Labelling of the Two Iron-binding Sites

The influence of pH changes upon the iron-binding properties of transferrin was investigated in the absence of chelating agents. The effects were demonstrated by spectrophotometry, gel filtration, and by studies of the intermolecular transfer of 59Fe from transferrin to conalbumin. At pH values below 6.7, diferric transferrin readily loses iron. The monoferric molecule, which is relatively resistant to acid dissociation, is preferentially formed. A temporary reduction of pH provides a simple method for selectively attaching iron to one metal-binding site, and allows double isotopic labelling of the transferrin molecule. This technique may permit further investigation of the physiological properties of the two iron-binding sites.     

Non-Transferrin Plasma Iron in β-Thalassaemia/Hb E and Haemoglobin H Diseases

Non-transferrin plasma iron concentrations were determined in 45 normal controls and in 37 patients with Hb H disease and 104 patients with β-thalassaemia/Hb E disease. This revealed that non-transferrin plasma iron exists in cases with severe iron overload, more striking in β-thalassaemia/Hb E than in Hb H disease. Non-transferrin plasma iron is associated with higher transferrin iron saturation and higher plasma ferritin levels. The most striking finding was the significantly higher non-transferrin plasma iron in splenectomized patients with β-thalassaemia/Hb E disease than in the non-splenectomized patients. In view of the potential toxicity of non-transferrin iron, this fraction of iron may be responsible for tissue damage in these patients especially after splenectomy.     

Effects of Inflammation on Iron and Transferrin Metabolism

S ummary . Experimentally induced acute inflammation in rats is associated with a fall in plasma iron and iron-binding capacity. Transferrin synthesis by the liver of these animals is normal and not increased in the presence of a low serum iron, as it is in iron deficiency.
Human splenic macrophages and rabbit pulmonary macrophages which were maintained in culture for up to 1 week showed an ability to take up protein-bound iron and labelled plasma proteins into the cytoplasm.
It is suggested that the macrophage may be the site both of iron sequestration and of transferrin degradation. This would provide a unified hypothesis to account for the finding of a low serum iron and TIBC in inflammatory disorders.     

Elevated non-transferrin bound iron in the lungs of patients with Pneumocystis carinii pneumonia

OBJECTIVE: The aim of the present work was to determine the concentrations of iron and iron-binding proteins in the lungs of patients suffering from Pneumocystis carinii (PCP), which is crucial for justifying the treatment with iron-chelating agents in this disease. PATIENTS AND METHODS: Bronchoalveolar lavage was performed in 10 HIV patients with PCP and five healthy controls. Total iron and iron-binding proteins (transferrin, ferritin and lactoferrin) were measured in acellular bronchoalveolar lavage fluid (BALF) in both groups. Iron was determined by atomic absorption spectrometry; transferrin and lactoferrin were measured using specific enzyme-linked immunosorbent assays (ELISA); and ferritin concentration was quantified by automated immunonephelometry. RESULTS: Our findings in patients with PCP demonstrated a six- to seven-fold increase of total iron levels and an eight-fold increase of ferritin in bronchoalveolar lavage fluid when compared with controls. No significant differences were found in transferrin or lactoferrin levels. Moreover, our results suggest that this iron is non-transferrin bound. CONCLUSION: Non-transferrin bound iron is increased in the lower respiratory tracts of PCP patients. This finding would lend experiment support to the use of iron-chelating agents in this disease.     

Functional heterogeneity of transferrin-bound iron: iron uptake by cell suspensions from bone marrow and liver and by cell cultures of fibroblasts and lymphoblasts.

According to the hypothesis of Fletcher and Huehns, functional differences exist between both iron-binding sites of transferrin. The site designated A should mainly be involved in the delivery of iron to erythroid cells, whereas site B should donate its iron preferentially to cells involved in the absorption and storage of iron. In the present study this hypothesis could be confirmed by in vitro experiments with various cell types. Iron transferrin preincubated with rat bone marrow cells donates less iron to rat bone marrow cells, Chinese hamster fibroblasts, human fibroblasts and human lymphoblasts than freshly prepared iron transferrin equal in iron and transferrin concentraion. Rat liver parenchymal cells, however, take up more iron from preincubated than from freshly prepared iron transferrin. Obviously, site A not only donates iron preferentially to erythroid cells but also to (rapidly) dividing nonerythroid cells in culture. From experiments with iron transferrin mixtures in which radioiron was present at low or high iron saturation, it could be concluded that rat bone marrow cells take up iron equally well from monoferric as from diferric transferrin. The observed functional heterogeneity could, therefore, not be ascribed to differences between monoferric and diferric transferrin.     

Iron metabolism in patients with different variants of pulmonary tuberculosis

Prior to treatment, 48 patients with different forms of pulmonary tuberculosis were examined. Serum iron concentrations, total iron-binding capacity of the serum (STIBC), its unsaturated iron-binding capacity (SUSIB), serum transferrin iron saturation coefficient (SC), total protein in the serum, red blood cells, hemoglobin, colour index were determined. All the parameters under study were in the normal range in patients with a favourable involutional course of pulmonary tuberculosis. In patients with acutely progressive pulmonary tuberculosis, serum iron levels, STIBC, SC were drastically decreased, while SUSIB was in the normal range. All this was attended by phenomena of hypochromic anemia. The pattern of the found changes leads to the conclusion that patients with acutely progressive tuberculosis develop iron-redistributing anemia caused by the changes in the amount and quality of transferrin, iron binding during free radical processes and mobilization of the antioxidant defense system rather than true iron deficiency.     

Bioavailability of carbonyl iron: a randomized, double-blind study

49 female blood donors with iron-deficiency anemia were treated with equal doses of iron either as carbonyl iron or ferrous sulfate in a randomized, double-blind fashion. The prevalence of side-effects was similar in the two groups. Mean values for hemoglobin concentration, mean corpuscular volume, corrected reticulocyte count, platelet count, serum iron, total iron-binding capacity, transferrin saturation or erythrocyte protoporphyrin did not differ significantly between the two groups throughout the study. After 16 weeks of therapy, the mean increase in hemoglobin iron was similar in both groups (p = 0.2). Estimates of net changes in total body iron suggested that the overall bioavailability of carbonyl iron was high, about 70% that of ferrous sulfate.     

Immunochemical Quantitation of Human Transferrin in Pregnancy and during the Administration of Oral Contraceptives

S ummary . The effects of pregnancy and oral contraceptive administration on serum transferrin levels were studied using an immunochemical method of estimation. Serum iron, total iron-binding capacity (TIBC) measured chemically, plasma bound iodine (PBI) and serum glutamic oxalacetic transaminase (SGOT) were also estimated on the same samples. The results show that the striking elevation in TIBC produced by oral contraceptives is due to increase in the transferrin level in the serum. The increase in serum transferrin, TIBC and PBI following the administration of oral contraceptives were of the same order as the changes produced physiologically during the second half of pregnancy. SGOT values were all normal. The findings suggest that changes in transferrin and other serum proteins produced by oral contraceptives are not due to liver damage but perhaps to increased protein synthesis. However, further study is necessary to elucidate the factors regulating the serum transferrin concentration and the effect on these of synthetic hormones.