Effect of transferrin, lactoferrin and chelated iron on human T-lymphocytes.

The effect of different forms of iron and iron-binding proteins on the proliferative response of human lymphocytes to phytohaemagglutinin (PHA) has been studied. Transferrin enhanced proliferation, the effect being proportional to the degree of iron saturation up to 100%, but decreased if additional iron was present. The lipophilic complex ferric pyridoxal isonicotinoyl hydrazone (FePIH) also enhanced proliferation, but the hydrophilic complex ferric nitrilotriacetate (FeNTA) was inhibitory. Fe-lactoferrin could not substitute for Fe-transferrin, although iron-free (apo) lactoferrin abrogated the inhibitory effect seen when iron levels exceed the binding capacity of transferrin. Lymphocyte ferritin levels increased 4-fold as the iron saturation of transferrin increased from 0 to 90% but no further increase was seen at higher iron levels, suggesting that lymphocytes are poorly equipped to detoxify excess iron through stimulation of ferritin synthesis. The effect of iron on the CD4:CD8 ratio after 72 h culture with PHA was also examined. The ratio was approximately 2:1 for cells cultured with transferrin at iron saturations between 0 and 75%, with FePIH, or without either, but decreased to 1.1:1 when cells were cultured in the presence of FeNTA, regardless of whether or not saturated Fe-transferrin was present. These results show that iron can affect lymphocyte proliferation and subset ratios in different ways according to the form and amount present, and may help to explain some of the immunological disturbances associated with iron overload.     

Maternal-foetal status of copper,iron, molybdenum,selenium and zinc in obese gestational diabetic pregnancies

Obesity is well known to be a contributory risk factor for several disease states, including diabetes mellitus. Paucity of data on maternal-foetal status of essential trace elements in obese diabetic pregnancies prompted us to undertake this study. Maternal venous and umbilical arterial and venous blood samples were collected from obese gestational diabetic patients (Body Mass Index (BMI) >30) and control obese pregnant women (BMI>30) at time of spontaneous delivery or caesarean sections and concentrations of essential trace elements such as Cu, Fe, Mo, Se and Zn were determined in various samples by atomic absorption spectrophotometry. Activities of antioxidant enzymes, superoxide dismutase (SOD), glutathione peroxidase (GPX) and total antioxidant (TAO) in maternal and umbilical blood were assessed using appropriate reagent kits. Maternal-foetal disposition and exchange parameters of elements studied were assessed using established criteria. Concentrations of Cu, Fe, Mo, Se and Zn in serum of control obese pregnant women (n=10) averaged 2404, 2663, 11.0, 89.0 and 666 μg/l respectively, while in the obese diabetic group (n=11), the corresponding values averaged 2441, 2580, 13.3, 85.1 and 610 μg/l respectively. Activities of antioxidant enzymes such as SOD, GPX and TAO were not significantly different in maternal veins of control and diabetic groups. Varying differences were noted in the case of antioxidant enzyme activities in umbilical blood samples of control and study groups. We conclude that obesity is not associated with significant alterations in antioxidant enzyme status in gestational diabetes and only with relatively minor alterations in status of some essential trace elements.     

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 μg/l), a statistical link was found between serum and erythrocyte ferritin levels. When the mothers had optimal iron status (serum ferritin ≥22 μg/l), 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/l). These data point to special iron kinetics during fetal life and the influence of maternal iron status.     

Lactoferrin-mediated transfer of iron to intracellular ferritin in human monocytes

Interactions of 125I-59Fe-lactoferrin with human monocytes were studied. After 4 hours of incubation, the uptake of 59Fe exceeded that of 125I. In dissociation studies the cellular 59Fe-activity was only partly dissociable during 16 h, whereas the 125I-activity could be released nearly completely. Disruption of the cells and studies on the cytosolic phase were performed employing gel-filtration and affinity chromatography. An appreciable amount of lactoferrin was found in the cytosolic phase. About 50% of the cytosolic 59Fe-activity was bound to ferritin. The results suggest that lactoferrin is internalized into monocytes and that iron is transferred to ferritin. These cellular events may contribute to an understanding of the accumulation of iron in the monocyte/macrophage system observed during inflammatory conditions.     

Ferroportin gene silencing induces iron retention and enhances ferritin synthesis in human macrophages

Missense mutations in the ferroportin gene (SLC11A3) result in haemochromatosis type 4 [HFE4, Online Mendelian Inheritance in Man (OMIM) reference 606069] or ferroportin disease, an autosomal dominant disorder characterized by predominantly reticuloendothelial iron accumulation. To verify whether HFE4 is caused by defective iron recycling because of loss of functionality of ferroportin, we down-regulated SLC11A gene expression in human macrophages by using small interfering RNAs (siRNAs). Transfection experiments with ferroportin siRNAs resulted in a marked reduction (about two-thirds on average) in ferroportin mRNA levels as detected by quantitative real time polymerase chain reaction. When macrophages were grown in medium supplemented with iron, cells transfected with siRNAs displayed three- to eightfold increases in staining intensities following Perls reaction. These macrophages also showed significant increases in H-ferritin content. The observation that ferroportin mRNA down-regulation to levels compatible with haplo-insufficiency causes increased iron retention and H-ferritin synthesis in cultured macrophages has important implications. First, this indicates that ferroportin levels must be finely regulated in order to maintain cellular iron homeostasis, and that both copies of SLC11A3 must function efficiently to prevent iron accumulation. Second, this observation supports the hypothesis that reticuloendothelial iron overload in patients with ferroportin disease is caused by loss-of-function mutations in the SLC11A3 gene that mainly impair macrophage iron recycling.     

Recent knowledge of the structure and function of lactoferrin and ferritin

A survey of recent knowledge on structure and importance of lactoferrin and ferritin is given. Lactoferrin is a symmetrically constructed glycoprotein which appears in the milk and in the body fluids and develops a bacteriostatic efficiency on account of the ability to Fe-binding together with other factors such as the IgA. In a particularly high concentration it is contained in the colostral milk of the woman. In the milk of the cattle the content is small. In the neutrophil granulocytes it is of importance for the functional ability in the phagocytosis. The ferritins are spherically constructed molecules which possess channels through which Fe-ions (up to 4,500 pro molecule) can be taken up or taken off. The ferritin content in the serum is correlated with the Fe-content of the liver and is dependent upon age as well as the Fe-supply. It decreases in Fe-deficiency: it increases in iron overload, in infectious diseases, in inflammation as well as in tumour development.     

Hepatic ferritin uptake and hepatic iron

The effect of hepatic iron on the uptake of ferritin was studied by perfusing livers from normal, iron-deficient and iron-loaded rats with 125I-labeled ferritin. Unlabeled ferritin with tracer doses of labeled ferritin in concentrations of 0.02 to 2,700 nmol/L were studied. Rats were made iron deficient by feeding an established iron-deficient diet for 3 wk. Rats were iron loaded by injection of iron dextran (50 mg/wk) for 3 wk. The mean percentage of uptake of ferritin was similar for doses ranging from 0.22 to 22.2 nmol/L of 125I-labeled ferritin. Uptake of ferritin in the normal animal was saturable, with an apparent maximal velocity of uptake of approximately 9.1 pmol/gm/min and a Michaelis-Menten constant of approximately 5 nmol/L at 37 degrees C. Uptake was minimal at 4 degrees C. The mean uptake of ferritin was 78% +/- 10% in the iron-deficient rats (mean hepatic iron = 1.5 mumol/gm), 79% +/- 10% in the normal animals (mean hepatic iron = 9.2 mumol/gm) and 78% +/- 8% in the iron-loaded animals (mean hepatic iron = 192 mumol/gm). In this experimental system, modulation of hepatic iron did not affect uptake of ferritin, suggesting that regulation of the hepatic ferritin receptor may not depend on hepatic iron content. The rapid uptake of ferritin by the liver despite iron overload is consistent with other observations of the nonregulation of non-transferrin-bound iron by hepatic iron and may play a role in the progressive iron overload seen in hemochromatosis.     

Evidence for a sequential transfer of iron amongst ferritin, transferrin and transferrin receptor during duodenal absorption of iron in rat and human

AIMTo elucidate the sequential transfer of iron amongst ferritin,transferrin and transferrin receptor under various iron status conditions.METHODSIncorporation of 59Fe into mucosal and luminal proteins was carried out in control WKY rats.The sequential transfer of iron amongst ferritin,transferrin and transferrin receptor was carried out in iron deficient,control and iron overloaded rats.The duodenal proteins were subjected to immunoprecipitation and quantitation by specific ELISA and in situ localization by microautoradiography and immunohistochemistry in tandem duodenal sections.Human duodenal biopsy(n = 36)collected from subjects with differing iron status were also stained for these proteins.RESULTSFerritin was identified as the major protein that incorporated iron in a time-dependent manner in the duodenal mucosa.The concentration of mucosal ferritin was significantly higher in the iron excess group compared to control,iron deficient groups(731.5 ± 191.96 vs 308.3 ± 123.36,731.5 ± 191.96 vs 256.0 ± 1.19,P ＜ 0.005),while that of luminal transferrin which was significantly higher than the mucosal did not differ among the groups(10.9 ± 7.6 vs 0.87 ± 0.79,11.1 ± 10.3 vs 0.80 ± 1.20,6.8 ± 4.7 vs 0.61 ± 0.63,P ＜ 0.001).In situ grading of proteins and iron,and their superimposition,suggested the occurrence of a sequential transfer of iron.This was demonstrated to occur through the initial binding of iron to luminal transferrin then to absorptive cell surface transferrin receptors.The staining intensity of these proteins varied according to the iron nutrition in humans,with intense staining of transferrin receptor observed in iron deficient subjects.CONCLUSIONIt is concluded that the intestine takes up iron through a sequential transfer involving interaction of luminal transferrin,transferrin-transferrin receptor and ferritin.     

The relationship between marrow iron stores, plasma ferritin concentrations and iron absorption.

The percentage absorption from a 3 mg dose of ferrous iron was measured in 50 subjects with iron stores that varied over a wide range. Iron status was assessed by a number of measurements, including the haemoglobin concentration, the plasma iron concentration, the total iron-binding capacity, the plasma ferritin concentration and the concentration of non-haem iron in the bone marrow. There were good inverse correlations between the log percentage iron absorption and both the log marrow non-haem iron concentration (r -0.94; P less than 0.001) and the log plasma ferritin concentration (r -0.78; P less than 0.001). In addition, there was a positive ferritin concentration (r +0.84; P less than 0.001). These results suggest that reticuloendothelial iron stores represent an important determinant of iron absorption and that their size can be guaged from the plasma ferritin concentration.     

Intake of essential trace elements (selenium, copper and iron) in the nutrition of patients hospitalized with liver cirrhosis

Dietary intake for three essential trace elements: selenium, copper and iron was studied in hospitalized patients receiving either ordinary meals or regimens adapted for liver cirrhosis. The aim of the study was to assess the effects of dietary manipulations: reduction in sodium intake for uncomplicated disease and protein restriction to 40 g per day in patients with hepatic encephalopathy. The meals proposed to these three groups were collected during fourteen days and daily intake for three elements was estimated by direct analysis of the assembled meals of the day. Dietary selenium was greatly affected by the restriction in protein intake contrary to copper and iron which were not significantly reduced. Moreover, overall daily trace element intake was rather low and clearly less than the most recent recommended allowances for these essential elements. Relationships between deficiency in some of these trace elements and worsening of the liver disease have been partly documented. They should encourage studies on the correction of dietary intake.     

Biliary excretion of iron and ferritin in idiopathic hemochromatosis

The role of biliary excretion of iron and ferritin in iron overload was studied and evaluated. Ten patients with idiopathic hemochromatosis and two groups of controls (14 gallstone patients and 16 healthy subjects) were included. Liver tissue (obtained by percutaneous or operative biopsy) was investigated with light microscopy and transmission electron microscopy in combination with x-ray microanalysis. Fasting bile samples were obtained through duodenal aspiration or at cholecystectomy. Iron was determined in liver tissue and bile using atomic absorption spectroscopy, and ferritin was determined in serum and bile with a radioimmunoassay technique. All patients with hemochromatosis had iron-positive staining as seen in light microscopy. Electron microscopy showed iron-containing proteins in the lysosomes and cytosol of liver parenchymal cells, and this observation was supported by x-ray microanalysis. Hepatic iron concentration was increased about eightfold in the patients with hemochromatosis (p less than 0.001). Biliary iron concentration, expressed per millimole of bile acid, was increased about twofold (p less than 0.05) and biliary ferritin concentration about fivefold (p less than 0.001) in hemochromatosis. Four of the patients with hemochromatosis were reexamined after completed treatment with venesection; this resulted in normalized biliary concentrations of iron and ferritin. We conclude that biliary secretion of ferritin occurs in humans and that both iron and ferritin excretion are enhanced in hepatic iron overload. The apparently limited capacity of biliary iron excretion may be of importance for the hepatic iron accumulation in hemochromatosis.     

Day-to-day variations in serum iron,serum iron binding capacity,serum ferritin and erythrocyte protoporphyrin concentrations in anaemic subjects

Day-to-day variations in serum iron, serum iron binding capacity, serum ferritin and erythrocyte protoporphyrin were determined on 2 successive days in 48 patients with anaemia. The correlation coefficients between the paired determinations were 0.86, 0.89, 0.95 and 0.95, and the day-to-day coefficients of variation (in per cent) were 33, 11, 12 and 13 for serum iron, serum iron binding capacity, erythrocyte protoporphyrin and serum ferritin, respectively. Thus, in patients with anaemia, day-to-day variations in serum iron, serum iron binding capacity, erythrocyte protoporphyrin and serum ferritin are at least as high as in healthy controls. The results indicate important limitations in the use, particularly, of serum iron in the clinical investigation of anaemia.     

Red cell ferritin and iron overload in heterozygous beta-thalassemia

Red cell ferritin was evaluated in 101 individuals with heterozygous beta-thalassemia to determine its clinical utility as an index for iron deficiency or overload in these subjects. The mean red cell ferritin for the total population was elevated threefold and showed a significant correlation with transferrin saturation, plasma ferritin, and HbA2 levels. Five of six subjects with reduced red cell ferritin had associated iron deficiency; a further five had iron deficiency and normal red cell ferritin. Normal red cell ferritin occurred in 51 subjects, and 44 had increased values. In the elevated red cell ferritin group, 21 individuals had associated normal plasma ferritin, and 23 had increased plasma ferritin. Only in the latter group was red cell ferritin significantly correlated with transferrin saturation and plasma ferritin. Ten individuals had a red cell ferritin greater than or equal to 150 attogram/cell, and liver biopsy performed in four showed grades II to IV iron overload. A clinical feature of subjects with both increased red cell and plasma ferritin levels was a high incidence of inappropriate iron administration. These findings suggest that red cell ferritin, particularly when combined with plasma ferritin, is a useful parameter for determining potential iron overload in individuals with heterozygous beta-thalassemia.