Plasmodium falciparum takes up iron from transferrin

Plasmodium falciparum, cultured in vitro, is inhibited by desferrioxamine, a specific iron chelating agent, despite the abundant haem iron which surrounds the parasite. In this report we show that the intraerythrocytic parasite obtains iron from transferrin. The parasite may therefore be freed of a requirement to catabolize haem to obtain iron thus minimizing haem toxicity.     

Inability to detect transferrin receptors on P. falciparum parasitized red cells

The mechanism by which P. falciparum takes up iron from transferrin has been explored. Binding of 125I labelled transferrin to parasitized red cells at 37 degrees C is two-fold greater than to control cells; at 0 degrees C there is no significant difference. The binding is non-specific as judged from the following: it is not saturable; it is not limited to transferrin as lactoferrin (which has iron binding domains) and bovine serum albumin (which does not) also bind in excess to parasitized red cells. A transferrin receptor complex could not be demonstrated when parasitized red cells, to which 125I transferrin was bound, were solubilized in Triton X100. Previous observation showed that uptake of transferrin iron by parasitized red cells is not accompanied by equimolar uptake of transferrin protein. We therefore suggest that nonspecifically bound transferrin is endocytosed, that the protein is degraded and the iron selectively retained.     

Antimalarial properties of orally active iron chelators

The appearance of widespread multiple drug resistance in human malaria has intensified the search for new antimalarial compounds. Metal chelators, especially those with high affinity for iron, represent one presently unexploited class of antimalarials. Unfortunately the use of previously identified chelators as antimalarials has been precluded by their toxicity and, in the case of desferrioxamine, the necessity for parenteral administration. The investigators now report that a new class of orally active iron chelators, namely the derivatives of alpha-ketohydroxypyridines (KHPs), are potent antimalarials against cultured Plasmodium falciparum. The KHPs evidently exert this effect by sequestering iron because a preformed chelator:iron complex has no antimalarial action. The pool(s) of iron being sequestered by the chelators have not been identified but may not include serum transferrin. Preincubation of human serum with KHPs followed by removal of the drug results in the removal of greater than 97% of total serum iron. Nonetheless, this serum effectively supports the growth of P falciparum cultures. Therefore the KHPs may exert antimalarial effect through chelation of erythrocytic rather than serum iron pool(s). The investigators conclude that these powerful, orally active iron chelators may form the basis of a new class of antimalarial drugs.     

Iron chelation with desferrioxamine B in adults with asymptomatic Plasmodium falciparum parasitemia.

To determine if iron chelation therapy has activity against human malaria, we administered desferrioxamine B in amounts of 100 mg/kg per day by continuous 72-hour subcutaneous infusions to 28 volunteers with asymptomatic Plasmodium falciparum infection in a randomized, double-blind, placebo-controlled crossover trial. Peripheral blood concentrations of P falciparum ring forms were determined at 12-hour intervals in all subjects and serum concentrations of desferrioxamine B + ferrioxamine (the iron complex of desferrioxamine B) were measured in 26 subjects. Geometric mean concentrations of asexual intraerythrocytic parasites decreased with both chelator and placebo treatment, but the decrement with desferrioxamine B was significantly greater than that with placebo (P less than .006) during both the initial and crossover periods. Compared with placebo, desferrioxamine B treatment was associated with an almost 10-fold enhancement of the rate of parasite clearance during both phases of the trial (P less than .007). Mean +/- SEM steady state concentrations of desferrioxamine B + ferrioxamine were 6.90 +/- 0.60 mumol/L at 36 hours and 7.72 +/- 0.68 mumol/L at 72 hours; in vitro, the ID50 has been reported to be approximately 4 to 20 mumol/L. No drug toxicity was detected. Parasitemia recurred in 19 of 24 participants followed-up over 1 to 6 months. We conclude that desferrioxamine B enhances the clearance of P falciparum parasitemia and that iron chelation may provide a new strategy to be developed for the treatment of malaria.     

The effect of iron chelation on haemopoiesis in MDS patients with transfusional iron overload

Long-term follow-up data are presented on changes in peripheral blood counts and Hb requirements of 11 patients with myelodysplastic syndromes (MDS) during iron chelation treatment with desferrioxamine for up to 60 months. The erythroid marrow activity was indirectly evaluated by repeated determinations of the serum transferrin receptor concentration. The efficacy of iron chelation was evaluated by repeated quantitative determination of the liver iron concentration by magnetic resonance imaging.
Reduction in the Hb requirement (≥50%) was seen in 7/11 (64%) patients. Five patients (46%) became blood transfusion independent. Platelet counts increased in 7/11 (64%) patients and the neutrophil counts in 7/9 (78%) evaluable patients. All patients in whom iron chelation was highly effective showed improvement of erythropoietic output accompanied by an increase in the serum transferrin receptor concentration.
It is concluded that reduction in cytopenia in MDS patients may be accomplished by treatment with desferrioxamine, if the iron chelation is efficient and the patients are treated for a sufficiently long period of time. Exactly how treatment with desferrioxamine works remains a challenge for further investigation.     

The effect of iron chelation on haemopoiesis in MDS patients with transfusional iron overload

Long-term follow-up data are presented on changes in peripheral blood counts and Hb requirements of 11 patients with myelodysplastic syndromes (MDS) during iron chelation treatment with desferrioxamine for up to 60 months. The erythroid marrow activity was indirectly evaluated by repeated determinations of the serum transferrin receptor concentration. The efficacy of iron chelation was evaluated by repeated quantitative determination of the liver iron concentration by magnetic resonance imaging. Reduction in the Hb requirement (≥50%) was seen in 7/11 (64%) patients. Five patients (46%) became blood transfusion independent. Platelet counts increased in 7/11 (64%) patients and the neutrophil counts in 7/9 (78%) evaluable patients. All patients in whom iron chelation was highly effective showed improvement of erythropoietic output accompanied by an increase in the serum transferrin receptor concentration. It is concluded that reduction in cytopenia in MDS patients may be accomplished by treatment with desferrioxamine, if the iron chelation is efficient and the patients are treated for a sufficiently long period of time. Exactly how treatment with desferrioxamine works remains a challenge for further investigation.     

Reversed siderophores act as antimalarial agents.

We describe here a family of biomimetic iron carriers that display high binding efficiency for ferric ions and favorable permeation properties across erythrocytic membranes. These carriers inhibit in vitro growth of Plasmodium falciparum by scavenging intracellular iron. The chemical features were realized by reproducing the iron-binding cavities of natural iron carriers (siderophores) and by systematic substitutions of their hydrophilic envelopes for more hydrophobic ones. In contrast to natural carriers, which participate in receptor-mediated iron uptake in cells and act as growth promoters, our synthetic carriers were designed to penetrate cellular membranes by diffusion, scavenge intracellular iron, and thereby act as growth inhibitors. Based on these properties we designate the compounds reversed siderophores and refer to the specific analogs of the natural ferrichrome as synthetic ferrichromes. The antimalarial activity of the synthetic ferrichromes correlated with their lipophilicity, and this antimalarial activity was averted when the chelators were applied as iron (III) complexes. The sites of synthetic ferrichrome action reside in the intraerythrocytic parasite and not in serum or on normal erythrocyte components. The agents were effective against all stages of parasite growth and against a variety of multidrug-resistant strains of P. falciparum. The most potent agent of this synthetic ferrichrome series, SF1-ileu, was not toxic to mammalian cells in culture and was 15-fold more potent and 20-fold faster acting than desferrioxamine. Taken in toto, these agents constitute a series of promising candidates for future use in malaria chemotherapy.     

Desferrioxamine therapy accelerates clearance of iron deposits after bone marrow transplantation for thalassaemia

We treated 18 heavily iron-loaded patients who had become ex-thalassaemics after bone marrow transplantation with subcutaneous desferrioxamine therapy for 5-20 months. As determined using serum ferritin concentration, transferrin saturation and stainable liver iron obtained in follow-up biopsies, marked decreases in body iron stores were observed with this regimen. Moreover, the liver function tests demonstrate a trend to normalization in all cases. Local skin reactions to desferrioxamine were the only toxicities observed. We conclude that pharmacological iron chelation is a safe and effective therapy in the reduction of iron deposits in this clinical situation; it therefore represents a valid alternative to phlebotomy in selected patients.     

Unrestricted growth of Plasmodium falciparum in microcytic erythrocytes in iron deficiency and thalassaemia

The mechanism(s) underlying the apparent resistance to malaria in certain inherited red cell disorders and iron deficiency anaemia remain poorly understood. The possibility that microcytic erythrocytes might inhibit parasite development, by physical restriction or reduced supply of nutrients, has been considered for many years, and never formally investigated. We sought to determine whether in vitro growth studies of P. falciparum could provide evidence to suggest that small red cell size contributes to malaria resistance in those red cell disorders in which microcytosis is a characteristic feature.
Invasion and development of P. falciparum in iron deficient red cells (mean values for mean cell volume [MCV] 66 fl, mean cell haemoglobin [MCH] 19 pg) and in the red cells of two gene deletion forms of α-thalassaemia (mean MCV 71 fl, MCH 22 pg) were normal, assessed both morphologically, and by ³H-hypoxanthine incorporation. Although parasite appearances were normal in all cell types, morphological abnormalities were noted in iron deficient and thalassaemic cells parasitized by mature stages of P. falciparum , notably cellular ballooning and extreme hypochromia of the red cell cytoplasm. Using electron microscopy, the red cell cytoplasm in parasitized thalassaemic cells showed reduced electron density and abnormal reticulation. Normal invasion rates were observed following schizogony in microcytic cells of both types.
Our findings indicate that whilst minor morphological abnormalities may be detected in parasitized iron deficiency and thalassaemic erythrocytes, development of P. falciparum in these conditions is not limited by small erythrocyte size.     

Expression of CD25 independently predicts early treatment failure of acute myeloid leukaemia (AML)

Iron is a critical and tightly regulated nutrient for both the malaria parasite and its human host. The importance of the relationship between host iron and the parasite has been underscored recently by studies showing that host iron supplementation may increase the risk of falciparum malaria. It is unclear what host iron sources the parasite is able to access. We developed a flow cytometry‐based method for measuring the labile iron pool (LIP) of parasitized erythrocytes using the nucleic acid dye STYO 61 and the iron sensitive dye, calcein acetoxymethyl ester (CA‐AM). This new approach enabled us to measure the LIP of P. falciparum through the course of its erythrocytic life cycle and in response to the addition of host serum iron sources. We found that the LIP increases as the malaria parasite develops from early ring to late schizont stage, and that the addition of either transferrin or ferric citrate to culture media increases the LIP of trophozoites. Our method for detecting the LIP within malaria parasitized RBCs provides evidence that the parasite is able to access serum iron sources as part of the host vs. parasite arms race for iron.     

The effect of iron overload and iron reductive treatment on the serum concentration of carbohydrate-deficient transferrin

Summary. The concentration of carbohydrate-deficient transferrin in serum (CDT) has been used as a reliable indicator of recent alcohol consumption. We have investigated the utility of this laboratory test in 20 patients with hereditary haemochromatosis (HH) by simultaneous evaluation of serum concentrations of liver transaminases, γ-glutamyl transpeptidase, iron, transferrin and asessment of the liver iron concentration by magnetic resonance imaging. 11 patients were re-examined during iron depletion with phelebotomies. In all 11 patients intensive but not maintenance iron removal was associated with an increase in serum CDT, in three patients even to levels above the reference range. The mean serum CDT increased from 8.5 (SD 2.2) U/1 to 16.6 (SD 7.2) U/1 (P < 0.001). Iron mobilization from the liver was found particularly responsible for the increase in serum CDT. Independent of this finding we found a significant semi-logarithmic correlation (r =−0.77. P = 0.009) between the MRI determined liver iron depletion. Our findings indicate that the utility of serum CDT as a measure of alcohol consumption in patients with HH may be compromised, especially during intensive iron depletion.     

The effects of short-term iron supplementation on iron status in infants in malaria-endemic areas

Iron deficiency and Plasmodium falciparum malaria are the two main causes of anemia in young children in region endemic for this disease. The impact on iron status of prophylactic oral iron supplementation (2 mg/kg/day from two to six months of age) and the duration of this effect were assessed in a group of 832 Tanzanian infants exposed to P. falciparum malaria. Iron parameters and red blood cell indices were assessed at 2, 5, 8, and 12 months of age. Infants who received iron supplements had a significantly lower prevalence of iron deficiency (P < 0.01 at 5 months and P < 0.001 at 8 and 12 months). Red blood cell indices (mean corpuscular volume, mean cell hemoglobin, and mean cell hemoglobin concentration) were increased in children receiving iron supplementation and they did not differ between those protected and unprotected against malaria. The prevalence of ferropenia was similar in children protected against malaria and in those who were not protected and did not receive iron supplements (34.7% versus 37.3% at 12 months of age). We concluded that iron supplementation between the ages of 2-6 months improves iron status at least up to 12 months of age. Malaria infection does not contribute to iron deficiency.     

Serum non-transferrin-bound iron in beta-thalassaemia major patients treated with desferrioxamine and L1

Non-transferrin-bound iron (NTBI) in plasma is toxic due to its ability to participate in free radical formation with resultant peroxidation and damage to cell membranes and other biomolecules. NTBI concentration was determined in serum in 12 normal volunteers and in 52 patients with beta-thalassaemia major by a modification of the method described by Singh et al (1990). There was no detectable NTBI in normal individuals. In the patients NTBI values ranged from -1.5 to 9.0 mumol/l (mean +/- SD: 3.6 +/- 2.3). The patients' serum ferritin concentrations ranged from 207 to 11,400 micrograms/l (2674 +/- 2538), total serum iron from 20 to 61 mumol/l (39.5 +/- 9.6) and transferrin saturation from 44 to 110% (84.5 +/- 13.8). The NTBI correlated significantly with serum ferritin (r = 0.467, P < 0.001), total serum iron (r = 0.608, P < 0.001) and transferrin saturation (r = 0.481, P < 0.005). When patients were grouped according to their compliance with desferrioxamine (DFX) therapy, the good compliers had significantly lower NTBI concentrations compared to the poor compliers (poor: 5.4 +/- 1.8 mumol/l v good: 2.7 +/- 1.7 mumol/l, P < 0.001). There was also a significant difference between the level of NTBI and whether or not the patients had complications of iron overload (5.2 +/- 1.7 mumol/l v 2.9 +/- 1.6 mumol/l, P < 0.001). During this study 10 patients were entered into a trial of the oral iron chelator 1,2- dimethyl-3-hydroxypyrid-4-one (L1). Their NTBI values were observed during the first 6 months of the trial and showed a significant fall (paired t-test: P = 0.007). These results suggest that the level of NTBI may prove helpful in assessing the efficiency of chelation in patients with transfusion dependent anaemia and help to predict organ damage.     

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.     

Assessment of antimalarial effect of ICL670A on in vitro cultures of Plasmodium falciparum

We tested in vitro the antimalarial properties of ICL670A, a newly developed iron chelator for the long-term oral treatment of iron overload. Ring-stage synchronized cultures of Plasmodium falciparum cultured in human erythrocytes were exposed to different concentrations of ICL670A and the conventional iron chelator, desferrioxamine B (DFO), for 48 h. Malarial growth was measured by incorporation of [3H]-hypoxanthine. ICL670A at 30 micromol/l had marked antimalarial activity that was observable by 6 h after beginning the exposure of ring-stage parasites to the agent. Over 48 h of culture, malarial growth was significantly lower with ICL670A than with DFO at concentrations of both 30 micromol/l (P = 0.008) and 60 micromol/l (P = 0.001). At 48 h, growth relative to control was 53% with ICL670A and 83% with DFO at concentrations of 30 micromol/l, and 20% with ICL670A and 26% with DFO at concentrations of 60 micromol/l. Standard 50% inhibitory concentrations (IC50s) were similar for ICL670A and DFO. Precomplexation with iron completely abolished the inhibitory effect of ICL670A, indicating that this new agent, like DFO, probably inhibits parasite growth via deprivation of iron from critical targets within the parasite. Further studies to address the question of the antimalarial potential of ICL670A in combination with classic antimalarials would be of interest.     

The effect of chelating agents on iron mobilization in Chang cell cultures.

The investigation of chelating agents with potential therapeutic value in patients with transfusional iron overload has been facilitated by the use of Chang cell cultures. These cells have been incubated with [59Fe]transferrin for 22 hr, following which most of the intracellular radioiron is found in the cytosol, distributed between a ferritin and a nonferritin form. Iron release from the cells depends on transferrin saturation in the medium, but when transferrin is 100% saturated, which normally does not allow iron release, desferrioxamine, 2,3-dihydroxybenzoic acid, rhodotorulic acid, cholythydroxamic acid, and tropolone all promote the mobilization of ferritin iron and its release from cells. They are effective to an approximately equal degree. The incubation of [59Fe]transferrin with tropolone in vitro at a molar ratio of 1:500 results in the transfer of most of the labeled iron to the chelator, reflecting the exceptionally high binding constant of this compound. How far these phenomena relate to therapeutic potentially remains to be seen.     

Inflammation and iron deficiency in the hypoferremia of obesity

CONTEXT: Obesity is associated with hypoferremia, but it is unclear if this condition is caused by insufficient iron stores or diminished iron availability related to inflammation-induced iron sequestration. OBJECTIVE: To examine the relationships between obesity, serum iron, measures of iron intake, iron stores and inflammation. We hypothesized that both inflammation-induced sequestration of iron and true iron deficiency were involved in the hypoferremia of obesity. DESIGN: Cross-sectional analysis of factors anticipated to affect serum iron. SETTING: Outpatient clinic visits. PATIENTS: Convenience sample of 234 obese and 172 non-obese adults. MAIN OUTCOME MEASURES: Relationships between serum iron, adiposity, and serum transferrin receptor, C-reactive protein, ferritin, and iron intake analyzed by analysis of covariance and multiple linear regression. RESULTS: Serum iron was lower (75.8+/-35.2 vs 86.5+/-34.2 g/dl, P=0.002), whereas transferrin receptor (22.6+/-7.1 vs 21.0+/-7.2 nmol/l, P=0.026), C-reactive protein (0.75+/-0.67 vs 0.34+/-0.67 mg/dl, P<0.0001) and ferritin (81.1+/-88.8 vs 57.6+/-88.7 microg/l, P=0.009) were higher in obese than non-obese subjects. Obese subjects had a higher prevalence of iron deficiency defined by serum iron (24.3%, confidence intervals (CI) 19.3-30.2 vs 15.7%, CI 11.0-21.9%, P=0.03) and transferrin receptor (26.9%, CI 21.6-33.0 vs 15.7%, CI 11.0-21.9%, P=0.0078) but not by ferritin (9.8%, CI 6.6-14.4 vs 9.3%, CI 5.7-14.7%, P=0.99). Transferrin receptor, ferritin and C-reactive protein contributed independently as predictors of serum iron. CONCLUSIONS: The hypoferremia of obesity appears to be explained both by true iron deficiency and by inflammatory-mediated functional iron deficiency.     

Early iron reduction programme for thalassaemia patients after bone marrow transplantation

Thirty thalassaemia patients received iron reduction starting at around 3 months post transplant. Sixteen received desferrioxamine and nine had phlebotomy, five patients had desferrioxamine followed by phlebotomy. The desferrioxamine group had higher serum ferritin levels at the start of iron reduction as compared to the phlebotomy group (5292 vs 2453 microg/l, P EQ 0.001). After 444 and 407 days of iron reduction, serum ferritins at cessation of iron reduction in both groups was similar (665 vs 588 microg/l). The rate of decline of serum ferritin in both groups was similar. There was no graft rejection during the programme. Early institution of iron reduction in ex-thalassaemia is safe.     

Defective iron supply for erythropoiesis and adequate endogenous erythropoietin production in the anemia associated with systemic-onset juvenile chronic arthritis

Systemic-onset juvenile chronic arthritis (SoJCA) is associated with high levels of circulating interleukin-6 (IL-6) and is frequently complicated by severe microcytic anemia whose pathogenesis is unclear. Therefore, we studied 20 consecutive SoJCA patients with hemoglobin (Hb) levels <12 g/dL, evaluating erythroid progenitor proliferation, endogenous erythropoietin production, body iron status, and iron supply for erythropoiesis. Hb concentrations ranged from 6.5 to 11.9 g/dL. Hb level was directly related to mean corpuscular volume (r = .82, P < .001) and inversely related to circulating transferrin receptor (r = - .81, P < .001) suggesting that the severity of anemia was directly proportional to the degree of iron-deficient erythropoiesis. Serum ferritin ranged from 18 to 1,660 microgram/L and was unrelated to Hb level. Bone marrow iron stores wore markedly reduced in the three children investigated, and they also showed increased serum transferrin receptor and normal-to-high serum ferritin. All 20 patients had elevated IL-6 levels and normal in vitro growth of erythroid progenitors. Endogenous erythropoietin (epo) production was appropriate for the degree of anemia as judged by both the observed to predicted log (serum epo) ratio 10.95 +/- 0.12) and a comparison of the serum epo- Hb regression found in these subjects with that of thalassemia patients. Multiple regression analysis showed that serum transferrin receptor was the parameter most closely related to hemoglobin concentration: variation in circulating transferrin receptor explained 61% of the variation in Hb level (P < .001). In 10 severely anemic patients, amelioration of anemia following intravenous iron administration resulted in normalization of serum transferrin receptor. Defective iron supply to the erythron rather than blunted epo production is the major cause of the microcytic anemia associated with SoJCA. A true body-iron deficiency caused by decreased iron absorption likely complicates long-lasting inflammation in the most anemic children, and this can be recognized by high serum transferrin receptor levels. Although oral iron is of no benefit, intravenous iron saccharate is a safe and effective means for improving iron availability for erythropoiesis and correcting this anemia. Thus, while chronically high endogenous IL-6 levels do not appear to blunt epo production, they are probably responsible for the observed abnormalities in iron metabolism. Anemia of chronic disease encompasses a variety of anemic conditions whose peculiar features may specifically correlate with the type of cytokine(s) predominantly released.     

Auxotrophs of Plasmodium falciparum dependent on p-aminobenzoic acid for growth.

The isolation of auxotrophic strains of a parasite offers new opportunities for studying parasitology. We have isolated cloned lines of Plasmodium falciparum that, unlike the parent line from which they were derived, rely on exogenous p-aminobenzoic acid (PABA) for growth. Isolation involved random mutagenesis of a cloned line of P. falciparum and subsequent selection of PABA-dependent parasites. Both parent and PABA-dependent clones were analyzed for PABA uptake and synthesis. Each clone takes up comparable amounts of PABA from the medium. The parent line, clone 3D7, can synthesize PABA de novo, whereas the PABA-dependent clones cannot. The requirement of exogenous PABA for growth by the auxotrophic strains coupled with their inability to synthesize PABA indicates that normal parasite growth can be completely supported by either synthesis or salvage. This work further clarifies the relationship between the availability of PABA and success of the parasite, an issue of debate from classic studies showing reduced parasite load in individuals on milk-fed diets.