Tissue Iron Stores Research Articles

Structure, function and clinical significance of transferrin receptors.

Iron plays an essential role in a spectrum of metabolic processes. Cellular iron uptake is facilitated by transferrin receptor (TfR)-mediated endocytosis. In recent years more insight has been obtained in TfR physiology and the regulation of cellular iron homeostasis. The synthesis of TfR and the iron storage protein ferritin is regulated reciprocally at the post-transcriptional level according to the cellular iron status. As a result of externalization of TfR during the endocytic cycle, a soluble form of TfR can be detected in serum. The serum TfR (sTfR) level is closely related to erythroid TfR turnover and the prime determinants of the sTfR concentration are cellular iron demands and erythroid proliferation rate. In the absence of a hyperplastic erythropoiesis the sTfR level is a sensitive parameter of early tissue iron deficiency. The entire spectrum of body iron status can be assessed by measurement of serum ferritin and sTfR levels, with ferritin as marker of tissue iron stores and sTfR as index of tissue iron needs. The sTfR may be a promising tool to detect iron deficiency in inflammatory states and in the anaemia of chronic disease as its concentration is, in contrast to ferritin levels, not influenced by the acute phase response. Determination of sTfR levels may also improve assessment of body iron stores during pregnancy and in neonates. Finally, the sTfR may be a useful parameter to monitor erythropoiesis in various clinical settings, for instance in the prediction of the haematological response to erythropoietin treatment. However, standardization of the sTfR assay, with definition of reference and pathological ranges, is necessary for the definitive introduction of the sTfR as major parameter of iron metabolism.

Abstracts of Literature

Abstracts of Literature

Body iron stores and the risk of carotid atherosclerosis: prospective results from the Bruneck study.

Fe2+ released from tissue iron stores may accelerate lipid peroxidation by virtue of its pro-oxidant properties and thus promote early atherogenesis. The present prospective survey addresses the potential association between serum ferritin concentrations and the 5-year progression of carotid atherosclerosis as assessed by ultrasonographic follow-up evaluations. The study population comprises a random sample of 826 men and women 40 to 79 years old. Serum ferritin was one of the strongest risk predictors of overall progression of atherosclerosis. The main part of this association appeared to act through modification of the atherogenic potential of LDL cholesterol (OR [95% CI] for a 1-SD unit increase in ferritin at LDL levels of 2.5, 3.6, and 4.9 mmol/L: 1.55 [1.30 to 1.85], 1.77 [1.40 to 2.24], and 2.05 [1.50 to 2.80]; P=.0012 for effect modification). Changes in iron stores during the follow-up period modified atherosclerosis risk, in that a lowering was beneficial and further iron accumulation exerted unfavorable effects. All these findings applied equally to incident atherosclerosis and the extension of preexisting atherosclerotic lesions. The significance of prominent iron stores in the development of carotid stenosis was clearly less pronounced. Finally, ferritin and LDL cholesterol showed a synergistic association with incident cardiovascular disease and death (n=59). The present study provided strong epidemiological evidence for a role of iron stores in early atherogenesis and suggests promotion of lipid peroxidation as the main underlying pathomechanism. This hypothesis could in part explain the sex difference in atherosclerotic vascular disease.

Purification and Characterization of Fet3 Protein, a Yeast Homologue of Ceruloplasmin

The FET3 gene product of Saccharomyces cerevisiae is an essential component of the high affinity iron transport system. Based on FET3 sequence homology to the multicopper oxidase family and iron oxidation studies in spheroplasts (De Silva, D. M., Askwith, C. C., Eide, D., and Kaplan, J. (1995) J. Biol. Chem. 270, 1098-1101), it was hypothesized that the Fet3 protein (Fet3p) was a cell surface ferroxidase. To further characterize the protein, we have isolated Fet3p from yeast membranes and purified the protein to apparent homogeneity. Consistent with its localization at the plasma membrane, Fet3p is a glycosylated protein. SDS-polyacrylamide gel electrophoresis analysis showed that the protein was present in two differentially glycosylated forms of approximately 120 and 100 kDa. Purified Fet3p is a copper-containing protein that is able to catalyze the oxidation of a variety of organic compounds in addition to ferrous iron. Azide and metal chelators strongly inhibited enzyme activity. Iron appeared to be the best substrate for the enzyme, and the apparent Km for ferrous oxidation was 2 microM. Interestingly, Fet3p was able to effectively catalyze the incorporation of iron onto apotransferrin. We conclude that Fet3p is a ferro-O2-oxidoreductase in yeast, homologous to the human plasma protein ceruloplasmin.

Hepatic hemosiderosis in non-human primates: quantification of liver iron using different field strengths.

Using a non-human primate model of idiopathic hemochromatosis, hemosiderin-induced T2 shortening of the liver was assessed at nine different field strengths over a range of 0.05 to 1.5 Tesla. The 1/T2 values increased linearly with field strength, with all specimens having approximately the same zero-field intercept. The slope of the field increase, termed "field-dependent T2 proton relaxation enhancement (PRE)", appeared to be proportional to the chemically determined tissue iron content, viz. 10.8 s-1T-1(mg Fe/g wet tissue)-1. The correlation between iron content and field-dependent T2 PRE (r = 0.94) was better than the correlation between iron content and 1/T2 values obtained at single field strengths. For livers containing > or = 2 mg Fe/g wet weight, biexponential T2 relaxation behavior emerged at higher field strengths, with the short T2 component (intracellular water) exhibiting a linear dependence of 1/T2 on field, while T2 of the long component (extracellular/sinusoidal water) was nearly field-independent. After maceration of the specimens, all T2 relaxation curves became monoexponential, including those for high iron content at high field strengths. The present data suggest that the use of double-field MR imaging to assess the field-dependent T2 PRE has potential for specific quantification of (liver) tissue iron stores.

Lipid peroxidation induced in vivo by hyperhomocysteinaemia in pigs

Much attention has been focused recently on the relationship between homocysteinaemia and the development of premature atherosclerosis. Hyperhomocysteinaemia constitutes as strong a risk factor for the development of the disease as either hypercholesterolaemia or smoking. Although the mechanism involved is unclear homocysteine exhibits prooxidative activity in vitro. This finding suggests that it may be involved in the oxidative modification of low density lipoprotein (LDL). In the current study hyperhomocysteinaemia was induced in eight domestic pigs by intermittent exposure to nitrous oxide for 4 weeks. At necropsy, cardiac tissue was removed and malondialdehyde (MDA) and the unsaturated fatty acid content were measured and compared with values obtained from air-breathing control animals. Nitrous oxide treated animals had significantly higher tissue concentrations of MDA than the controls. There was also a reduction in the contribution of linoleic and linolenic acids to the total fatty acid content of heart. The hyperhomocysteinaemic animals also had a significantly higher iron concentration in the heart than controls. Hyperhomocysteinaemia was associated with elevations in tissue iron stores and increased in vivo lipid peroxidation.

Erythrocyte ferritin in human neonates: maternofetal iron kinetics revisited.

Serum and erythrocyte ferritin concentrations were measured in 369 healthy term neonates and their mothers, with a view to understanding maternofetal iron kinetics. Erythrocyte ferritin concentrations in the neonates were on average 100 times higher than maternal values, while those of serum ferritin were about 10 times higher. Maternal and fetal iron status were correlated in the overall study population. Serum and erythrocyte ferritin values were also correlated with each other in both the mothers and neonates. To study the influence of iron status on these conditions, we formed three groups of mother-child pairs of equal size, based on maternal iron status (serum ferritin). The correlations found in the overall population were not always present in each subgroup. In the infants born to mothers with adequate tissue iron reserves (serum ferritin > 12 micrograms/I), a statistical link was found between serum and erythrocyte ferritin levels. When the mothers had optimal iron status (serum ferritin > or = 22 micrograms/I), there was a correlation between the neonatal and maternal erythrocyte ferritin levels. No such links were found when the mothers had low tissue iron stores (serum ferritin < 12 mg/I). These data point to special iron kinetics during fetal life and the influence of maternal iron status.

Oral iron chelation with HBED

Iron chelation therapy with desferrioxamine (DFO) has dramatically improved the outlook in β-thalassemia. Parenteral DFO reduces tissue iron stores, prevents iron-induced organ damage, and reduces morbidity and mortality, with little serious toxicity. However, the burden of prolonged subcutaneous portable pump infusions, high cost, and patient noncompliance have prompted the development of new methods of administration and new formulations of DFO as well as oral iron chelators. Deferiprone (L1), the only oral iron chelator studied in large long-term clinical trials, is less effective and more toxic than DFO and may not adequately control iron overload; however, compliance and quality of life are improved. Combinations of two iron chelators (such as parenteral DFO plus oral L1, or 2,3-DHB; or oral L1 plus HBED) have been shown to produce additive and synergistic effects, explained by the shuttle hypothesis. Iron bound to a “shuttle”—an oral agent that mobilizes tissue iron—is exchanged in the bloodstream with a “sink”— such as parenteral DFO—and excreted via the kidneys, while the shuttle is reutilized. Combination therapy may produce enhanced iron excretion, target specific iron compartments, minimize side effects, increase treatment options, improve compliance, and facilitate individualization of therapy. Better understanding of the kinetics of iron metabolism, iron overload, and chelation should improve therapeutic strategies.

Arthropathy in thalassaemia patients receiving deferiprone

The iron chelator deferiprone (L1) reduces tissue iron stores in iron-loaded patients. Three of sixteen patients treated with deferiprone developed joint pain and swelling without evidence of systemic lupus erythematosus (SLE). Articular cartilage, synovial hypertrophy and iron deposition, and synovial lining cell proliferation, with no inflammatory or allergic reaction, were observed on synovial exploration and biopsy. Symptoms resolved partly or completely during continued drug administration. We hypothesise that deferiprone-induced shifts of iron to synovium resulted in tissue damage, accelerated by free-radical formation during incomplete complexation of iron and this bidentate chelator. This deferiprone-associated symptom complex is not associated with drug-induced SLE, and does not progress in severity during continued therapy.

Field dependent transverse relaxation rate increase may be a specific measure of tissue iron stores

The degree to which MRI magnet field strength affects measured transverse relaxation rates (R2) defines a measure termed the field dependent R2 increase (FDRI). We report here the results of in vivo and in vitro experiments that were conducted to evaluate whether FDRI is a potentially useful measure of tissue iron stores. T2 relaxation times were obtained using two clinical MRI instruments operating at 0.5 and 1.5 Tesla, and relaxation rates (R2) were calculated as the reciprocal of T2. The in vivo experiment measured R2 in human brain frontal white matter, caudate nucleus, putamen, and globus pallidus. The FDRI was very highly correlated with published brain iron levels for the four regions examined. The in vitro experiment measured R2 in agarose gel-based phantoms containing physiologic forms and amounts proteins involved in iron storage and transport (ferritin, apoferritin, transferrin, and apotransferrin). Significant field dependence was observed only for the ferritin phantoms. The differences in the R2 values obtained at the two field strengths were striking, and were proportional to the ferritin levels of the phantoms. These studies suggest that FDRI may be a specific measure of tissue ferritin. The quantitative significance of the results to imaging and possible applications to the clinical investigation of pathologic states are discussed.

Iron stores and serum transferrin receptor levels during recombinant human erythropoietin treatment of anemia in rheumatoid arthritis.

Ten rheumatoid arthritis (RA) patients with anemia of chronic disorders (ACD) were treated with recombinant human erythropoietin (r-Hu-Epo) using a dose of 250 U/kg s.c. 3 times a week for 6 weeks, in order to evaluate its effects on the anemia, iron stores, and serum-soluble transferrin receptor (sTfR) levels. All patients showed a rise in hemoglobin (Hb). Median Hb increased from 5.9 (5.5-7.0) at baseline to 6.7 (5.8-7.8) at 3 weeks and to 7.2 (5.9-8.5) mmol/l at 6 weeks during treatment. Ferritin levels decreased significantly during the 6 weeks, and five patients were iron deficient after 6 weeks of treatment. TfR levels increased significantly at 3 and 6 weeks during treatment. These preliminary findings may indicate that r-Hu-Epo is effective in improving ACD in RA. The sTfR rise may be explained by an increase in erythroid precursor cell mass or increased TfR expression and a decrease in tissue iron stores, although direct effects of Epo on TfR regulation cannot be excluded. Large double-blind studies with r-Hu-Epo in patients with RA and ACD are warranted.

Reduction of tissue iron stores and normalization of serum ferritin during treatment with the oral iron chelator L1 in thalassemia intermedia

In patients with thalassemia intermedia in whom hyperabsorption of iron may result in serious organ dysfunction, an orally effective iron-chelating drug would have major therapeutic advantages, especially for the many patients with thalassemia intermedia in the Third World. We report reduction in tissue iron stores and normalization of serum ferritin concentration after 9-month therapy with the oral chelator 1,2-dimethyl-3-hydroxypyrid-4-one (L1) in a 29-year-old man with thalassemia intermedia and clinically significant iron overload (SF 2,174 micrograms/L, transferrin saturation 100%; elevated AST and ALT, abnormal cardiac radionuclide angiogram) who was enrolled in the study with L1 75 mg/kg/day after he refused deferoxamine therapy. L1-Induced 24-hour urinary iron excretion during the first 6 months of therapy was (mean +/- SD, range) 53 +/- 30 (11 to 109) mg (0.77 mg/kg), declining during the last 3 months of L1 to 24 +/- 14 (13-40) mg (0.36 mg/kg), as serum ferritin decreased steadily to normal range (present value, 251 micrograms/L). Dramatic improvement in signal intensity of the liver and mild improvement in that of the heart was shown by comparison of T1-weighted spin echo magnetic resonance imaging with images obtained immediately before L1 administration was observed after 9 months of L1 therapy. Hepatic iron concentration decreased from 14.6 mg/g dry weight of liver before L1 therapy to 1.9 mg/g liver after 9 months of therapy. This constitutes the first report of normalization of serum ferritin concentration in parallel with demonstrated reduction in tissue iron stores as a result of treatment with L1. Use of L1 as a therapeutic option in patients with thalassemia intermedia and iron overload appears warranted.

Reduction of Tissue Iron Stores and Normalization of Serum Ferritin During Treatment With the Oral Iron Chelator L1 in Thalassemia Intermedia

Abstract In patients with thalassemia intermedia in whom hyperabsorption of iron may result in serious organ dysfunction, an orally effective iron- chelating drug would have major therapeutic advantages, especially for the many patients with thalassemia intermedia in the Third World. We report reduction in tissue iron stores and normalization of serum ferritin concentration after 9-month therapy with the oral chelator 1,2- dimethyl-3-hydroxypyrid-4-one (L1) in a 29-year-old man with thalassemia intermedia and clinically significant iron overload (SF 2,174 micrograms/L, transferrin saturation 100%; elevated AST and ALT, abnormal cardiac radionuclide angiogram) who was enrolled in the study with L1 75 mg/kg/day after he refused deferoxamine therapy. L1-Induced 24-hour urinary iron excretion during the first 6 months of therapy was (mean +/- SD, range) 53 +/- 30 (11 to 109) mg (0.77 mg/kg), declining during the last 3 months of L1 to 24 +/- 14 (13–40) mg (0.36 mg/kg), as serum ferritin decreased steadily to normal range (present value, 251 micrograms/L). Dramatic improvement in signal intensity of the liver and mild improvement in that of the heart was shown by comparison of T1- weighted spin echo magnetic resonance imaging with images obtained immediately before L1 administration was observed after 9 months of L1 therapy. Hepatic iron concentration decreased from 14.6 mg/g dry weight of liver before L1 therapy to 1.9 mg/g liver after 9 months of therapy. This constitutes the first report of normalization of serum ferritin concentration in parallel with demonstrated reduction in tissue iron stores as a result of treatment with L1. Use of L1 as a therapeutic option in patients with thalassemia intermedia and iron overload appears warranted.

The Use of Nuclear Magnetic Resonance Imaging in Monitoring Total Body Iron in Hemodialysis Patients With Hemosiderosis Treated With Erythropoietin and Phlebotomy

Two hemodialysis patients with hemosiderosis were treated with combined erythropoietin and repeated phlebotomy. Serial nuclear magnetic resonance (NMR) imaging and serum ferritin levels were used to monitor the efficacy of treatment. This treatment modality has definite advantages over chronic deferoxamine therapy. NMR image-derived parameters offer an objective, accurate, and noninvasive indication of tissue iron stores.

Iron Supplementation After Femoral Head Replacement for Patients With Normal Iron Stores

To assess the efficacy of oral iron therapy in the recovery of patients' hemoglobin levels after major surgery. Randomized controlled trial. Private orthopedic practice confined to one large community hospital. One hundred seventy consecutive elderly patients undergoing hip surgery; 75 failed to meet entry hematologic or medical criteria; 95 were randomized, with 16 withdrawn because of complications. Thirty-seven patients received ferrous sulfate orally four times a day for the duration of their hospitalization. Forty-two patients who received no iron supplement served as the control group. Changes in hemoglobin levels and reticulocyte counts over the 2- to 3-week follow-up period. There was no significant difference in mean hemoglobin levels between the treatment and control groups (95% confidence interval [CI] for difference of -6.6 to 5.4 g/L). Corrected reticulocyte fractions increased equally in both groups (95% CI for difference of -9 x 10(3) to 2 x 10(-3). The study was designed to detect a difference in mean hemoglobin levels of 8.5 g/L or greater or a difference in mean reticulocyte fraction of 10 x 10(-3) between the two groups with a power of 0.80 at the .05 (two-sided) level of significance. The administration of oral iron supplements to elderly, healthy orthopedic patients postoperatively did not hasten the recovery of hemoglobin levels, provided adequate tissue iron stores were present.

The Impact of Varying Degrees of Iron Nutriture on Several Functional Consequences of Iron Deficiency in Rats

The impact of varying severities of iron-deficiency anemia on fasting blood glucose, plasma triiodothyronine, heart norepinephrine concentrations and resting oxygen consumption were evaluated. Male weanling Sprague-Dawley rats were assigned to one of six dietary groups (4, 6, 11, 16, 23 or 40 mg Fe/kg diet) for 6 wk. Hemoglobin, liver iron and transferrin saturation were significantly lower in the 4 and 6 mg Fe/kg diet groups relative to the other groups and were indicative of anemia, low tissue iron stores and impaired erythropoiesis. Fasting blood glucose and heart norepinephrine concentrations were significantly higher and lower, respectively, in the 4 and 6 mg Fe/kg diet groups than the three highest dietary Fe groups. Although fasting blood glucose was significantly inversely correlated (r = -0.89, P = 0.0001) with hemoglobin concentration; a significant quadratic relationship (R 2 = 0.70, P = 0.0001) existed between hemoglobin and heart norepinephrine concentration. Differences in plasma triiodothyronine concentrations and resting oxygen consumption were not significant among the groups. Thus, base on hemoglobin concentration as an index of the severity of iron deficiency, these findings demonstrate that certain physiological manifestations of iron deficiency occur at even moderate-to-mild degrees of anemia.

Abnormal vaginal bleeding secondary to iron deficiency in a thirteen year old

Abnormal vaginal bleeding in perimenarchal females is usually attributed to immaturity of the hypothalmic/pituitary/gonadal axis (i.e., dysfunctional uterine bleeding). Iron deficiency as a cause for abnormal bleeding has been described, but is poorly understood. This case report describes a 13-year-old Hispanic female with iron deficiency anemia as a presumptive cause for two episodes of abnormal vaginal bleeding. Anemia may be a cause as well as an effect of menorrhagia in young teenagers. Work-up should include evaluation of tissue iron stores if other etiologies for abnormal bleeding are not found.

Noninvasive assessment of tissue iron stores.

Multivariate regression methods were used to create two equations for calculation of the "iron index," a predictor of bone marrow iron scores. With the use of the most efficient cut-off point, approximately 90% of 210 patients could be diagnosed correctly. Selection of a criterion that minimizes false negatives (missed iron deficiency) would increase the number of unnecessary bone marrow aspirations, but, even then, more than a third of such tests could be avoided. Rapid turnaround time would be desirable for all measurements used to calculate the iron index, although it is recognized that for most hospitalized patients the length of hospital stay is determined by severity of an underlying disease, not the presence of anemia.

Effects of iron deficiency on endurance and muscle enzyme activity in man

The purpose of the present study was to evaluate the effects of iron deficiency on enzyme activities and endurance. Iron deficiency was induced in 9 healthy male subjects by repeated venesections. After a period of 9 wk (range, 8-11 wk) when the subjects had become iron deficient as defined by laboratory parameters, blood was retransfused to reestablish the control hemoglobin concentration. In this state it was possible to evaluate the effect of iron deficiency isolated from anemia. In samples secured by muscle biopsies, glycolytic, oxidative, and iron depending enzymes were analyzed in the control (C) and anemic (A) states and after retransfusion (R). There were no significant changes in the maximal activities of any of the enzymes studied. The capillary/fiber ratio remained unchanged between C (1.92) and R (1.94). Times to exhaustion on treadmill tests were 49 min, 11 s in C, 26 min, 33 s in A, and 52 min, 3 s in R. Vo2max was 4.55 1 X min-1 in C, 3.74 1 X min-1 in A, and 4.45 1 X min-1 in R. An artificially induced iron deficiency defined by conventional laboratory parameters did not affect endurance when transfusion of red blood cells was performed in order to exclude the influence of a low hemoglobin concentration. A 4-wk period of severely depleted or absent tissue iron stores did not affect the maximal activities of various enzymes in human skeletal muscle.

Role of iron in Trypanosoma cruzi infection of mice.

The role of iron in experimental infection of mice with Trypanosoma cruzi was investigated. B6 mice had a transient parasitemia and a transient anemia, both of maximal intensity 28 d after the inoculation of T. cruzi. There was a biphasic hypoferremic host response to infection with T. cruzi with the peak hypoferremia also occurring 28 d after inoculation of the parasite. The mortality rate from infection was increased from 23% in phosphate-buffered saline-treated B6 mice to 50% in a group of B6 mice receiving iron-dextran (P less than or equal to 0.025), whereas depletion of iron stores with the iron chelator desferrioxamine B and an iron-deficient diet provided complete protection of B6 mice (P less than or equal to 0.05). The mortality rate in the highly susceptible C3H strain was reduced from 100% in the control group to 45% (P less than or equal to 0.025) in the iron-depleted group. The tissue iron stores were altered in mice receiving either iron-dextran or desferrioxamine B and an iron-deficient diet. In vitro, T. cruzi was shown to require both a heme and a nonheme iron source for an optimal growth rate. The effects of iron excess or depletion on the outcome of infection with T. cruzi correlated both with the growth requirements of the parasite for iron and with the availability of intracellular iron. Thus, it was suggested that the hypoferremic response, by sequestering iron within intracellular stores, potentially enhanced the pathogenicity of the intracellular parasites. Furthermore, the in vivo effects of iron excess and depletion correlated with an effect of iron on the growth rate and pathogenicity of the parasite.