Non-transferrin-bound iron in plasma following administration of oral iron drugs

Non-transferrin-bound iron (NTBI) was detected in serum samples from volunteers with normal iron stores or from patients with iron deficiency anaemia after oral application of pharmaceutical iron preparations. Following a 100 mg ferrous iron dosage, NTBI values up to 9 μM were found within the time period of 1–4 h after administration whereas transferrin saturation was clearly below 100%. Smaller iron dosages (10 and 30 mg) gave lower but still measurable NTBI values. The physiological relevance of this finding for patients under iron medication has to be elucidated.     

Non-transferrin-bound iron uptake by rat liver. Role of membrane potential difference

Non-transferrin-bound iron is efficiently cleared from serum by the liver and may be primarily responsible for the hepatic damage seen in iron-overload states. We tested the hypothesis that transport of ionic iron is driven by the negative electrical potential difference across the liver cell membrane. Extraction of 55Fe-labeled ferrous iron (1 microM) from Krebs bicarbonate buffer by the perfused rat liver was continuously monitored as the transmembrane potential difference (measured using conventional microelectrodes) was altered over the physiologic range by isosmotic ion substitution. Resting membrane potential in Krebs bicarbonate buffer was -28 +/- 1 mV. Perfusion with 1 microM ferrous iron caused a reversible 3 +/- 1 mV depolarization, and higher concentrations of iron caused even greater depolarization. Conversely, depolarization of the liver cells consistently reduced iron extraction. Replacement of sodium with potassium (70 mM) or choline (131 mM) depolarized the hepatocytes to -15 and -20 mV and decreased iron extraction by 28 and 31%, respectively. Perfusion with bicarbonate-free solutions containing tricine buffer (10 mM) reduced the membrane potential to -23 mV and reduced iron extraction by 18%. In contrast, the high basal extraction of iron (91.1 +/- 1.4%) was not further increased by substitution of nitrate for chloride (-46 mV) or infusion of glucagon (-34 mV). All effects were reversible, suggesting that perfusion with 1 microM iron produced little toxicity. These findings are consistent with an electrogenic transport mechanism for uptake of non-transferrin-bound iron that is driven by the transmembrane potential difference.     

Non-transferrin-bound iron uptake in Belgrade and normal rat erythroid cells

Belgrade (b) rats have an autosomal recessive, microcytic, hypochromic anemia. Transferrin (Tf)-dependent iron uptake is defective because of a mutation in DMT1 (Nramp2), blocking endosomal iron efflux. This experiment of nature permits the present study to address whether the mutation also affects non-Tf-bound iron (NTBI) uptake and to use NTBI uptake compared to Tf-Fe utilization to increase understanding of the phenotype of the b mutation. The distribution of ⁵⁹Fe²⁺ into intact erythroid cells and cytosolic, stromal, heme, and nonheme fractions was different after NTBI uptake vs. Tf-Fe uptake, with the former exhibiting less iron into heme but more into stromal and nonheme fractions. Both reticulocytes and erythrocytes exhibit NTBI uptake. Only reticulocytes had heme incorporation after NTBI uptake. Properly normalized, incorporation into b/b heme was ∼20% of +/b, a decrease similar to that for Tf-Fe utilization. NTBI uptake into heme was inhibited by bafilomycin A1, concanamycin, NH₄Cl, or chloroquine, consistent with the endosomal location of the transporter; cellular uptake was uninhibited. NTBI uptake was unaffected after removal of Tf receptors by Pronase or depletion of endogenous Tf. Concentration dependence revealed that NTBI uptake into cells, cytosol, stroma, and the nonheme fraction had an apparent low affinity for iron; heme incorporation behaved like a high-affinity process, as did an expression assay for DMT1. DMT1 serves in both apparent high-affinity NTBI membrane transport and the exit of iron from the endosome during Tf delivery of iron in rat reticulocytes; the low-affinity membrane transporter, however, exhibits little dependence on DMT1. J. Cell. Physiol. 178:349–358, 1999. © 1999 Wiley-Liss, Inc.     

Iron and cadmium uptake by duodenum of hypotransferrinaemic mice

Absorption from food is an important route for entry of the toxic metal, cadmium, into the body. Both cadmium and iron are believed to be taken up by duodenal enterocytes via the iron regulated, proton-coupled transporter, DMT1. This means that cadmium uptake could be enhanced in conditions where iron absorption is increased. We measured pH dependent uptake of ¹⁰⁹Cd and ⁵⁹Fe by duodenum from mice with an in vitro method. Mice with experimental (hypoxia, iron deficiency) or hereditary (hypotransferrinaemia) increased iron absorption were studied. All three groups of mice showed increased ⁵⁹Fe uptake (p<0.05) compared to their respective controls. Hypotransferrinaemic and iron deficient mice exhibited an increase in ¹⁰⁹Cd uptake (p<0.05). Cadmium uptake was not, however, increased by lowering the medium pH from 7.4 to 6. In contrast, ⁵⁹Fe uptake (from ⁵⁹FeNTA₂) and ferric reductase activity was increased by lowering medium pH in control and iron deficient mice (p<0.05). The data show that duodenal cadmium uptake can be increased by hereditary iron overload conditions. The uptake is not, however, altered by lowering medium pH suggesting that DMT1-independent uptake pathways may operate.     

Non-transferrin-bound iron in plasma or serum from patients with idiopathic hemochromatosis. Characterization by high performance liquid chromatography and nuclear magnetic resonance spectroscopy

The nature of non-transferrin-bound iron in the plasma or serum of iron-overloaded hemochromatosis patients was studied by high performance liquid chromatography (HPLC) and high resolution nuclear magnetic resonance (NMR). 500-MHz proton Hahn spin-echo NMR spectra of plasma or serum, combined with the use of the iron chelator desferrioxamine, suggests complexation of iron ions with citrate and a possible involvement of acetate. Addition of FeCl3 to hemochromatosis samples broadened the NMR signals from citrate. HPLC analysis rigorously confirmed the presence of an iron-citrate complex in ultrafiltrates of plasma or serum studies with added FeCl3 or desferrioxamine supported this conclusion. It is proposed that non-transferrin-bound iron in the plasma of iron-overloaded patients exists largely as complexes with citrate and possibly also as ternary iron-citrate-acetate complexes. The presence of such complexes would account for the ability of non-transferrin-bound iron to be measurable by the bleomycin assay and for its rapid clearance from the circulation by the liver.     

Uptake of 26-Al and 67-Ga into brain and other tissues of normal and hypotransferrinaemic mice

Aluminium uptake from blood into tissues of control and homozygous hypotransferrinaemic (hpx/hpx) mice, following continuous intravenous infusion of Al and Ga, has been compared with that of gallium, a proposed tracer for aluminium. Al uptake into tissues of control (hpx/+ and +/+) mice occurred in the order (expressed as a space): bone 464.7ml 100g; renal cortex 102.9ml 100g; liver 13.0ml 100g; spleen 8.4ml 100g and brain 0.8ml 100g. Ga uptakes were similar in liver, spleen and brain, but smaller in the renal cortex and bone, at one-third and one-fifth of the values for Al, respectively. In the hypotransferrinaemic mice, uptake of Ga into all tissues was increased, especially in renal cortex (ninefold) and bone (twentyfold) as compared with the controls. Increases in Ga uptakes into cerebral hemisphere, cerebellum and brain stem of the hypotransferrinaemic mice were 3.8, 4.2 and 2.8 fold, respectively. Al uptake into tissues of the hypotransferrinaemic mice was similar to control values except in bone where it was three times greater. Pre-treatment of control animals with the anti-transferrin receptor antibody, RI7 208, enhanced Ga uptake in all tissues, the effect being greatest in renal cortex (tenfold) and bone (ninefold). Ga uptakes into cerebral hemisphere, cerebellum and brain stem in the mice pre-treated with RI7 208 were 6.4, 6 and 10 times greater than in untreated mice, respectively. No influence of antibody on Al uptake into mouse tissues was observed except in spleen where it was three times greater than in untreated mice. Hence, transport of aluminium and gallium into mouse tissues is not similar under all conditions. Non-transferrin mediated transport of each metal can occur into all tissues, especially in renal cortex and bone, where gallium may be a suitable marker for aluminium.     

Iron metabolism in Trypanosoma lewisi infection: serum iron and serum iron-binding capacity.

Progressive changes in iron levels, total iron binding capacity and hematocrit values in sera of rats infected with Trypanosoma lewisi are described. The host dietary group were: (1) complete or full complement; (2) iron-deficient, and (3) pair-fed or calorically restricted. The hematocrit values of T. lewisi-infected rats given the various diets were not significantly different from those of the controls. The decrease in total iron binding capacity (TIBC) of rats inoculated with T. lewisi and fed complete and pair-fed diets ranged up to 15% over uninfected controls. TIBC levels in rats fed an iron-deficient diet and inoculated with T. lewisi ranged up to 32% over uninfected controls. TIBC levels of deficient infected rats were significantly different from the controls from day 90 to infection to the end of the observation period. Serum iron (SI) values of non-infected rats regardless of dietary regimen showed significantly higher values than T. lewisi-infected animals between days 95 and 120. The average SI value, for this period, in adequately fed control rats was 204 +/- 7 microgram/100 ml as compared to 172 +/- 5 microgram/100 for trypanosome-infected rats. SI levels of rats on a pair-fed diet and infected with T. lewisi decreased to 17% over uninfected controls. SI levels of animals on an iron-deficient diet and infected with T. lewisi decreased up to 76% over uninfected controls.     

Radioprotective effects of serum thymic factor in mice.

Serum thymic factor (FTS) reduced mortality of mice after total-body irradiation with 7.56 Gy X rays. The radioprotective effect was achieved by daily repeated subcutaneous injections of 3-100 micrograms FTS, while doses higher than 300 micrograms/day/mouse were neither radioprotective nor toxic. Similarly, degeneration of the spleen was moderated by 3-100 micrograms FTS but not by 500 micrograms FTS in sublethally (3.78 Gy) irradiated mice. Histological examination showed that hematopoiesis was enhanced in the spleen by daily injections of 10 micrograms FTS. Spleen cells from the FTS-treated mice incorporated more [3H]thymidine in culture with or without concanavalin A. The treatment with FTS increased the production of colony-stimulating factor in the spleen as well as in peritoneal macrophage-like cells, and caused a significant increase in the number of granulocyte-macrophage colony-forming cells both in the spleen and in the femoral bone marrow. Furthermore, FTS prevented a decrease in circulating neutrophils in the sublethally irradiated mice. Prominent overshoot recovery of myelopoiesis, which occurred occasionally in sublethally irradiated mice, did not occur in the FTS-treated mice. The decrease in blood erythrocytes was also significantly reduced. These observations imply that this thymic hormone has potential as a radioprotector.     

Comparison of serum iron, total iron binding capacity, ferritin, and percent transferrin saturation in nine species of apparently healthy captive lemurs

Lemurs kept in captivity have been reported to be highly prone to accumulate excessive amounts of iron in tissues (hemosiderosis). Diagnosis of the condition is most commonly made during a postmortem examination because an antemortem diagnosis requires a liver biopsy, a procedure that may not be well tolerated by all animals. The lack of a noninvasive method to evaluate iron status in captive lemurs limits investigators' ability to effectively screen animals for the presence of hemosiderosis, and to detect the condition early when treatment protocols are most effective. This study was conducted in an effort to provide data regarding iron analyte values in healthy captive lemurs of multiple species. The relationship of various iron-related metabolites was evaluated in 177 clinically normal lemurs of nine different species. Serum iron (sI), total iron binding capacity (TIBC), and ferritin concentration were measured directly and the percent transferrin saturation (TS) was calculated. Significant differences in various iron metabolites were observed among several species, suggesting that normal reference values for iron metabolites in lemurs may need to be developed on a species by species basis.     

EPR study of serum ceruloplasmin and iron transferrin in myocardial infarction.

Electron paramagnetic resonance (EPR) analysis of frozen serum from myocardial infarction patients has been conducted. Signal at g=4.3 was found definitively attributable to iron(III)-transferrin complex. Imcrease of serum ceruloplasmin as compared to normal was confirmed, with a concomitant decrease of iron-transferrin content. A mechanism for such correlated variation is hypothesized.     

Variations in serum calcium between strains of inbred mice.

Serum calcium concentrations of C3H/Fg, C3H/HeJFg, AKR/Fg, A/Fg, and LCS/Fg mice were compared at 4 and 7 mo of age. In the C3H/Fg and A-strain mice, calcium levels did not differ significantly between the 2 strains nor between the 2 age groups. The C3H/HeJFg, LCS/Fg, and C57BL/Fg strains formed a distinct group with similar calcium levels, but at a significantly higher level than C3H/Fg and A/Fg group. Again there was no significant difference between the 4- and 7-mo groups. The AKR/Fg strain was distinct from both groups in that higher calcium levels characteristic of the second group (C3H/HeJFg, LCS/Fg, and C57BL/Fg, were seen at 4 mo of age, but lower calcium levels similar to those of the C3H/Fg and A/Fg strains were found at 7 mo of age.