The effect of apotransferrin on iron release from Caco-2 cells, an intestinal epithelial cell line

The Caco-2 cell line grown in bicameral chambers was used to study the effect of transferrin in the basal chamber on the transepithelial transport of iron. We have shown that when iron was offered as 59Fe on the apical surface of the Caco-2 cells, transport of 59Fe into the basal chamber was stimulated by 50 micromol/L apotransferrin. Here, we examined the effect on 59Fe transport of lower concentrations of apotransferrin, as well as the effects on transport of ovo-, cobalt-, and ferri-transferrin and of iron chelators with an affinity for iron greater than that of transferrin. The stimulation of 59Fe transport was more sensitive to the presence of apotransferrin with a Km of 0.078 +/- 0.008 micromol/L compared with ferri-transferrin with a Km of 1.24 +/- 0.39 micromol/L (P < .006). 59Fe transport was less sensitive to diethylenetriaminopenta-acetic acid (DTPA) than apotransferrin with Kms of 1.52 +/- 0.70. The chelator nitrilotriacetic acid (NTA) exhibited no stimulation of 59Fe transport. Analysis of laser scanning confocal micrographs showed that apotransferrin labeled with Texas Red is internalized by Caco-2 cells from the basal side and localizes in distinct vesicles above the nucleus. The sensitivity of apotransferrin in stimulating Fe transport suggests a unique interaction of apotransferrin with the basal surface of the intestinal epithelium.     

Iron mobilisation and cellular protection by a new synthetic chelator O-Trensox

We tested a new synthetic, 8-hydroxyquinoline-based, hexadentate iron chelator, O-Trensox and compared it with desferrioxamine B (DFO). Iron mobilisation was evaluated: (i) in vitro by using ferritin and haemosiderin; DFO mobilised iron much more rapidly from ferritin at pH 7.4 than did O-Trensox, whereas at pH 4, ferritin and haemosiderin iron mobilisation was very similar with both chelators; (ii) in vitro by using cultured rat hepatocytes which had been loaded with 55Fe-ferritin; here DFO was slightly more effective after 100 hr than O-Trensox; (iii) in vivo administration i.p. to rats which had been iron-loaded with iron dextran; O-Trensox mobilised 51.5% of hepatic iron over two weeks compared to 48.8% for DFO. We also demonstrated the effect of O-Trensox in decreasing the entry of 55Fe citrate into hepatocyte cultures. The protective effect of O-Trensox against iron toxicity induced in hepatocyte cultures by ferric citrate was shown by decreased release of the enzymes lactate dehydrogenase (LDH), aspartate aminotransferase (AST), and alanine aminotranferase (ALT) from the cultures and, using electron paramagnetic resonance (EPR) measurements, decreased production of lipid radicals. O-Trensox was more effective than DFO in quenching hydroxyl radicals in an acellular system.     

Pyridoxal isonicotinoyl hydrazone and its analogs: potential orally effective iron-chelating agents for the treatment of iron overload disease

At present, the only iron (Fe) chelator in clinical use for the treatment of Fe overload disease is the tris-hydroxamate deferoxamine (DFO). However, DFO suffers from a number of disadvantages, including the need for subcutaneous infusion (12 to 24 hours a day, 5 or 6 times per week), its poor intestinal absorption, and high cost. Therefore, there is an urgent need for an efficient, economical, and orally effective Fe chelator. Pyridoxal isonicotinoyl hydrazone (PIH) is a tridentate Fe-chelating agent that shows high Fe chelation efficacy both in vitro in cell culture models and also in vivo in rats and mice. In addition, this chelator is relatively nontoxic, economical to synthesize, and orally effective, and it shows high selectivity and affinity for Fe. However, over the last 10 years the development of PIH and its analogs has largely been ignored because of justifiable interest in other ligands such as 1,2-dimethyl-3-hydroxypyrid-4-one (L1). Unfortunately, recent clinical trials have shown that significant complications occur with L1 therapy, and it is controversial whether this chelator is effective at reducing hepatic Fe levels in patients. Because of the current lack of a clinically useful Fe chelator to replace DFO, PIH and its analogs appear to be potential candidate compounds that warrant further investigation. In this review we will discuss the studies that have been performed to characterize these chelators at the chemical and biologic levels as effective agents for treating Fe overload. The evidence from the literature suggests that these ligands deserve further careful investigation as potential orally effective Fe chelators.     

Receptor-mediated recognition and uptake of iron from human transferrin by Staphylococcus aureus and Staphylococcus epidermidis

Staphylococcus aureus and Staphylococcus epidermidis both recognize and bind the human iron-transporting glycoprotein, transferrin, via a 42-kDa cell surface protein receptor. In an iron-deficient medium, staphylococcal growth can be promoted by the addition of human diferric transferrin but not human apotransferrin. To determine whether the staphylococcal transferrin receptor is involved in the removal of iron from transferrin, we employed 6 M urea-polyacrylamide gel electrophoresis, which separates human transferrin into four forms (diferric, monoferric N-lobe, and monoferric C-lobe transferrin and apotransferrin). S. aureus and S. epidermidis but not Staphylococcus saprophyticus (which lacks the transferrin receptor) converted diferric human transferrin into its apotransferrin form within 30 min. During conversion, iron was removed sequentially from the N lobe and then from the C lobe. Metabolic poisons such as sodium azide and nigericin inhibited the release of iron from human transferrin, indicating that it is an energy-requiring process. To demonstrate that this process is receptor rather than siderophore mediated, we incubated (i) washed staphylococcal cells and (ii) the staphylococcal siderophore, staphyloferrin A, with porcine transferrin, a transferrin species which does not bind to the staphylococcal receptor. While staphyloferrin A removed iron from both human and porcine transferrins, neither S. aureus nor S. epidermidis cells could promote the release of iron from porcine transferrin. In competition binding assays, both native and recombinant N-lobe fragments of human transferrin as well as a naturally occurring human transferrin variant with a mutation in the C-lobe blocked binding of 125I-labelled transferrin. Furthermore, the staphylococci removed iron efficiently from the iron-loaded N-lobe fragment of human transferrin. These data demonstrate that the staphylococci efficiently remove iron from transferrin via a receptor-mediated process and provide evidence to suggest that there is a primary receptor recognition site on the N-lobe of human transferrin.     

Pyoverdin is essential for virulence of Pseudomonas aeruginosa

The role of pyoverdin, the main siderophore in iron-gathering capacity produced by Pseudomonas aeruginosa, in bacterial growth in vivo is controversial, although iron is important for virulence. To determine the ability of pyoverdin to compete for iron with the human iron-binding protein transferrin, wild-type P. aeruginosa ATCC 15692 (PAO1 strain) and PAO pyoverdin-deficient mutants were grown at 37 degrees C in bicarbonate-containing succinate medium to which apotransferrin had been added. Growth of the pyoverdin-deficient mutants was fully inhibited compared with that of the wild type but was restored when pyoverdin was added to the medium. Moreover, when growth took place at a temperature at which no pyoverdin production occurred (43 degrees C), the wild-type PAO1 strain behaved the same as the pyoverdin-deficient mutants, with growth inhibited by apotransferrin in the presence of bicarbonate and restored by pyoverdin supplementation. Growth inhibition was never observed in bicarbonate-free succinate medium, whatever the strain and the temperature for growth. In vivo, in contrast to results obtained with the wild-type strain, pyoverdin-deficient mutants demonstrated no virulence when injected at 10(2) CFU into burned mice. However, virulence was restored when purified pyoverdin originating from the wild-type strain was supplemented during the infection. These results strongly suggest that pyoverdin competes directly with transferrin for iron and that it is an essential element for in vivo iron gathering and virulence expression in P. aeruginosa. Rapid removal of iron from [59Fe]ferritransferrin by pyoverdin in vitro supports this view.     

Transferrin, iron, and serum lipids enhance or inhibit Mycobacterium avium replication in human macrophages

Mycobacterium avium grows exponentially over 7 days in human macrophages when they are cultured in serumless medium. Normal serum inhibits this replication. When serum lipids were extracted using chloroform, the inhibitor was present in the lipid-free component. The lipid extract significantly enhanced M. avium replication. Iron (Fe2+) added at 8-80 micrograms/mL to infected macrophage cultures in serum resulted in enhanced mycobacterial replication. Serum-induced inhibition of bacterial growth in serumless medium could be duplicated with apotransferrin at 50-500 micrograms/mL. At 1000 micrograms/mL, apotransferrin no longer inhibited bacterial growth. Holotransferrin was not inhibitory, and at 500 micrograms/mL, it enhanced M. avium growth. Depletion of the transferrin in serum by affinity chromatography using goat anti-transferrin on protein G-Sepharose removed inhibitory activity. These results indicate that transferrin levels, transferrin saturation, iron levels, and serum lipids can profoundly alter the replication of M. avium in association with macrophages.     

Iron uptake in Ustilago maydis: tracking the iron path

In this study, we monitored and compared the uptake of iron in the fungus Ustilago maydis by using biomimetic siderophore analogs of ferrichrome, the fungal native siderophore, and ferrioxamine B (FOB), a xenosiderophore. Ferrichrome-iron was taken up at a higher rate than FOB-iron. Unlike ferrichrome-mediated uptake, FOB-mediated iron transport involved an extracellular reduction mechanism. By using fluorescently labeled siderophore analogs, we monitored the time course, as well as the localization, of iron uptake processes within the fungal cells. A fluorescently labeled ferrichrome analog, B9-lissamine rhodamine B, which does not exhibit fluorescence quenching upon iron binding, was used to monitor the entry of the compounds into the fungal cells. The fluorescence was found intracellularly 4 h after the application and later was found concentrated in two to three vesicles within each cell. The fluorescence of the fluorescently labeled FOB analog CAT18, which is quenched by iron, was visualized around the cell membrane after 4 h of incubation with the ferrated (nonfluorescent) compounds. This fluorescence intensity increased with time, demonstrating fungal iron uptake from the siderophores, which remained extracellular. We here introduce the use of fluorescent biomimetic siderophores as tools to directly track and discriminate between different pathways of iron uptake in cells.     

Desferrioxamine and vitamin E protect against iron and MPTP-induced neurodegeneration in mice

To elucidate the neuroprotective effects of the iron chelator desferrioxamine (DFO) and the antioxidant vitamin E on excessive iron-induced free radical damage, a chronic iron-loaded mice model was established. The relationship between striatal iron content, oxidized to reduced glutathione ratio, hydroxyl radical (.OH) levels and dopamine concentrations were observed in DFO or vitamin E pretreated iron-loaded/1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated C57BL/6 mice. The results demonstrated that both DFO and vitamin E inhibit the iron accumulation and thus reverses the increase in oxidized glutathione (GSSG), oxidized to reduced glutathione ratios, .OH and lipid peroxidation levels. The striatal dopamine concentration was elevated to normal value. Our data suggested that: (1) iron may induce neuronal damage and thus excessive iron in the brain may contribute to the neuronal loss in PD; (2) iron chelators and antioxidants may serve as potential therapeutic agents in retarding the progression of neurodegeneration.     

Attenuation of cochlear damage from noise trauma by an iron chelator, a free radical scavenger and glial cell line-derived neurotrophic factor in vivo

Tissue injury by reactive oxygen species (ROS) may play a role in noise-induced hearing loss (NIHL). Since iron is involved in ROS generation, we studied if an iron chelator, deferoxamine mesylate (DFO), alone or in combination with mannitol, a hydroxyl scavenger and weak iron chelator, attenuates NIHL. Further, we investigated if glial cell line-derived neurotrophic factor (GDNF) provides additive or synergistic protection of the cochlea from acoustic trauma when given together with DFO and mannitol. Pigmented female guinea pigs were exposed to noise (4 kHz octave band, 115 dB SPL, 5 h). One hour before, immediately after, and 5 h after noise exposure, subjects received an injection of 5 ml saline/kg (control, group I), 100 mg DFO/kg (group II), 15 mg mannitol/kg (group III), or both DFO and mannitol (group IV and V). Animals in group V underwent implantation of an osmotic pump filled with GDNF (100 ng/ml) in the left ear 4 days before noise. Each treatment afforded some protection from noise damage. Group I showed significantly greater outer hair cell loss and threshold shifts at two or more frequencies compared to groups II through V. GDNF provided an additive functional, but not morphological, protection with DFO and mannitol. These findings indicate that iron chelators can attenuate NIHL, as do ROS scavengers, supporting the notion that ROS generation plays a role in NIHL. Additional functional protection provided with GDNF suggests that GDNF may attenuate noise-induced cochlear damage through a mechanism that is additive with antioxidants.     

Aspirin and platelets: the antiplatelet action of aspirin and its role in thrombosis treatment and prophylaxis

The aim of this study was to investigate the influence of iron present in the growth medium of Staphylococcus aureus on the bacterial adhesion to collagen. The experiments were extended to determinate the siderophore production and to examine the S. aureus isolates surface hydrophobicity. The addition of iron to metal deficient defined medium causes the change in hydrophobicity of the examined S. aureus strains surfaces from hydrophilic to hydrophobic. The presence of iron in staphylococcal growth medium alters also the adhesion to the surface covered with collagen. Four out of six S. aureus strains adhere to collagen weaker when cells come from iron-rich medium. Majority of tested strains produce markedly less of siderophores in media containing the excess of iron (1 and 10 microM Fe) and there is no staphylococcal siderophore activity in the growth medium with a very high concentration of this compound (120 microM Fe). The obtained results indicate that the iron-stressed conditions influence the staphylococcal adhesion to collagen.     

Ferritin in cultured human cytotrophoblasts: synthesis and subunit distribution

The present study aims at the role of ferritin in the regulation of syncytiotrophoblast free iron levels. The differentiated cytotrophoblast cell in culture is used as a model for this maternal-fetal interface. Cytotrophoblast cells isolated from term placentae are cultured in iron-poor (Medium 199), iron-depleted [desferrioxamine(DFO)] and iron-supplemented [diferric transferrin (hTF-2Fe), ferric ammonium citrate (FAC)] medium. Distribution and de novo synthesis of isoferritins is studied, together with the cellular iron concentration and the ferritin iron saturation. Compared to ferritin isolated from total placenta, ferritin obtained from villous tissue is enriched with acidic isoforms. This observation is in agreement with measured light (L) to heavy (H) subunit ratios < 1 of de novo synthesized ferritin in cultured cytotrophoblast cells. Neither iron-poor culture medium, nor hTf-2Fe supplemented medium affects the cellular iron or ferritin concentration. FAC increased the cellular ferritin iron saturation and (by synthesis) the acidic isoferritin concentrations. The results strongly suggest, that the term syncytiotrophoblast is able to balance transferrin-mediated iron uptake and iron release. In case of FAC supplementation, the syncytiotrophoblast is unable to keep intracellular iron low, and ferritin synthesis is stimulated. The predominance of acidic ferritins and the preferential synthesis of H subunits can be functionally explained by the established fact that iron incorporation in acidic ferritins is faster due to the presence of ferroxidase centres. Damage by free iron catalysed hydroxyl radical formation is therefore minimized.     

Effects of agents that inhibit cellular iron incorporation on bladder cancer cell proliferation

Agents that interfere with cellular iron (Fe) incorporation inhibit tumor cell proliferation, including metals that bind to transferrin (Tf) such as gallium (Ga) or indium (In) and Fe chelators such as desferrioxamine (DFO). Ga nitrate is effective in the treatment of metastatic bladder cancer and these patients exhibit evidence for interference with Fe metabolism. We show here that bladder cancer cell proliferation in vitro is dependent on Tf-Fe. Concentrations of DFO that can be readily achieved in vivo inhibit cellular proliferation even in the presence of physiologic concentrations of Tf-Fe. Inhibition of proliferation by Tf-Ga is associated with decreased cellular Fe incorporation. However, when a physiologic concentration of Tf-Fe is added to an equimolar concentration of Tf-Ga, significant Fe incorporation is evident despite inhibition of proliferation. Thus, besides interference with Fe incorporation, Ga may also interfere with intracellular Fe distribution and/or directly inhibit an Fe- (or non-Fe-) requiring process necessary for cellular proliferation. DFO followed sequentially by Tf-Ga results in marked potentiation of inhibition of proliferation. The effects of this combination appear to be related to both interference with Fe metabolism and increased Ga uptake. This sequential combination may be useful in the treatment of bladder cancer.     

Influence of vitamin E on preventive or therapeutic effect of MFA and DTPA in cadmium toxicity

Exposure to hypochlorous acid (HOCl), the main product of the reaction of neutrophil myeloperoxidase (MPO), H2O2, and Cl-, reportedly decreases apotransferrin's iron binding capacity. Optimal transferrin iron binding requires the coexistent binding of anions such as bicarbonate (HCO3-) near the protein's two iron binding sites. Recently, we found that if HCO3- was also present during HOCl exposure, apotransferrin retained its ability to inhibit iron-catalyzed hydroxyl radical generation. Therefore, we examined apotransferrin iron binding capacity after exposure to the MPO/H2O2/I- system in the presence and absence of several anions (HCO3-, H2PO4, SO4(2-), and ClO4-) known to bind to apotransferrin. Although the MPO system decreased apotransferrin iron uptake to only 46% of the untreated apotransferrin control, apotransferrin treated in the presence of 1 mM HCO3- or H2PO4- retained 84 and 74%, respectively, of its iron binding capacity. Similar results were seen when apotransferrin was treated with NaOCl. These results could not be explained on the basis of a loss of MPO activity or scavenging of HOCl. In contrast, SO4(2-) and ClO4- were unable to prevent the MPO-mediated loss of apotransferrin iron binding capacity. NaOCl had no effect on the ability of transferrin to bind any of these anions, as assessed by the anion-induced change in apotransferrin absorbance spectrum. HCO3- but not H2PO4-, SO4(2-), or ClO4- decreased MPO-mediated oxidation (iodination) of apotransferrin. Under some conditions H2PO4- actually increased apotransferrin iodination. HCO3- and H2PO4- may protect apotransferrin from MPO-mediated oxidative damage by preventing selective oxidation of one or both iron binding sites. This process may allow transferrin to retain its iron binding function during MPO exposure in vivo.     

P507+N235双溶剂萃取除铁生产实践

介绍了在硫酸介质中使用P507+N235双溶剂萃取体系萃取除铁的工艺应用。通过生产实践发现,铁以三价态被萃取,有机相由15%P507+5%N235+80%260#稀释剂组成,相比2∶1,铁萃取率达到98%以上,在反萃剂为250g/L稀硫酸溶液,相比4∶1的条件下反萃,铁反萃率达到98%以上,反萃液经均相渗析膜分离回收酸,渗析残液通过控制pH,可采用铁矾法、中和除铁和砷酸铁等工艺除铁,铁脱除率均可达到90%以上。     

Uptake of iron from transferrin by isolated rat-liver mitochondria mediated by phosphate compounds

1. Isolated rat-liver mitochondria accumulate iron from transferrin at neutral pH by a mechanism which is markedly stimulated by small-molecular-weight polyphosphate compounds. The efficiency of the phosphate compounds decreases in the order: pyrophosphate > ATP > GTP > 2,3-bis-(phospho)glycerate > phosphate. 2. The uptake has a very low energy dependence, and it does not depend on the hydrolysis of ATP or the saturation of transferrin, but it increases in parallel to the concentration of iron to reach a saturation level of 800-1200 pmol iron/mg protein. 3. Following a chase with unlabelled transferrin (125I)-labelled transferrin bound to the mitochondria remains constant, whereas the progressive uptake of 59Fe levels off. 4. During reincubation of iron-loaded mitochondria up to 30% of the iron is mobilized in the presence of ascorbate and ATP (or apotransferrin). 5. The results suggest that iron is mobilized from transferrin by the polyphosphate compounds outside the mitochondria in a subsequent reaction.     

Uptake and processing of 59Fe-labelled and 125I-labelled rat transferrin by early organogenesis rat conceptuses in vitro

The delivery of iron to the early organogenesis rat embryo has been studied, using 59Fe- and 125I-labelled rat transferrin. Rat conceptuses at 9.5 days postconception were cultured for 27 or 51 h in whole rat serum. Rat transferrin labelled with 59Fe was added for the final 0.1, 0.5, 6, 24 or 48 h of culture. Radioactivity accumulated progressively in both the embryo and the visceral yolk sac. Similar results were obtained when unconjugated 59Fe3+ was added to the rat serum used as culture medium. Both acid-soluble and acid-insoluble 59Fe were substantially present in the embryo and yolk sac after all exposure periods. When conceptuses were cultured in the presence of 125I-labelled rat transferrin, acid-soluble radioactivity was progressively released into the culture medium, but accumulation into the embryo and visceral yolk sac was slight and did not change with duration of exposure to the labelled protein. Similar findings were obtained using 125I-labelled bovine serum albumin. In these experiments, there was a close correspondence between the amount of iron accumulated by the embryo and visceral yolk sac in the final 24 h of a 51-h culture and the amount of transferrin converted into acid-soluble products in the same period. Visceral yolk sacs from 17.5-day pregnant rats were explanted and cultured in the presence of 59Fe-labelled rat transferrin, 125I-labelled rat transferrin or 125I-labelled bovine serum albumin, for periods up to 3 h. Again uptake of 59Fe increased with time of incubation, and the 125I-labelled proteins were digested to acid-soluble products which were released into the culture medium. The results indicate that transferrin delivers iron for incorporation into both the embryo and the visceral yolk sac, and are consistent with a mechanism involving receptor-mediated endocytosis of iron-laden transferrin by the cells of the visceral yolk sac. The transferrin itself appears to be quantitatively degraded, following delivery of iron to the yolk sac cells, a result that differs from findings in other cell types, in which the protein is not degraded but returns to the plasma membrane to participate in further cycles of iron acquisition and delivery.     

'Oversaturation' of transferrin after intravenous ferric gluconate (Ferrlecit(R)) in haemodialysis patients

BACKGROUND: Chronic haemodialysis causes blood loss and iron-deficiency. This can be corrected with intravenous preparations, e.g. sodium ferric-gluconate (FeGl). In two patents complaints of hypotension and malaise during FeGl infusion coincided with high levels of serum iron and a calculated transferrin iron saturation above 100%. Iron toxicity could be the cause of these complaints. Free iron is known to aggravate the toxicity of free radicals and other reactive oxygen products that are constantly formed in the body. We compared four rates of FeGl infusion with regard to iron parameters. METHODS: 20 dialysis patients received a total of 26 infusions of FeGl. A rapid infusion of 135 mg (Protocol A (n=10)) or 62.5 mg (Protocol B (n=7)) of FeGl was given during the last 30 min of dialysis. A slow infusion of 125 mg (Protocol C (n=9)) or 62.5 mg (Protocol D (n=10)) was given during 4 or 4.5 h of dialysis. Blood was taken at regular intervals, before, during, and after dialysis for determination of serum iron, transferrin, ferritin, haematocrit, total protein, albumin, and lactate dehydrogenase (LDH). Transferrin saturation was calculated from transferrin and serum iron. RESULTS: With rapid infusion A (125 mg) the highest levels of serum iron (median 120 (range 40-159) micromol/l) and transferrin saturation (207 (84-331)%) were seen at the end of the infusion. These were significantly higher than the peak levels with B, C, and D (P相似文献   

HBED: the continuing development of a potential alternative to deferoxamine for iron-chelating therapy

To further examine the potential clinical usefulness of the hexadentate phenolic aminocarboxylate iron chelator N, N'-bis(2-hydroxybenzyl)ethylenediamine-N,N'-diacetic acid (HBED) for the chronic treatment of transfusional iron overload, we performed a subchronic toxicity study of the HBED monosodium salt in rodents and have evaluated the iron excretion in primates induced by HBED. The HBED-induced iron excretion was determined for the monohydrochloride dihydrate that was first dissolved in a 0.1-mmol/L sodium phosphate buffer at pH 7.6 and administered to the primates either orally (PO) at a dose of 324 micromol/kg (149.3 mg/kg, n = 5), subcutaneously (sc) at a dose of 81 micromol/kg (37.3 mg/kg, n = 5), sc at 324 micromol/kg (n = 5), and sc at 162 micromol/kg (74.7 mg/kg) for 2 consecutive days for a total dose of 324 micromol/kg (n = 3). In addition, the monosodium salt of HBED in saline was administered to the monkeys sc at a single dose of 150 micromol/kg (64.9 mg/kg, n = 5) or at a dose of 75 micromol/kg every other day for three doses, for a total dose of 225 micromol/kg (n = 4). For comparative purposes, we have also administered deferoxamine (DFO) PO and sc in aqueous solution at a dose of 300 micromol/kg (200 mg/kg). In the iron-loaded Cebus apella monkey, whereas the PO administration of DFO or HBED even at a dose of 300 to 324 micromol/kg was ineffective, the sc injection of HBED in buffer or its monosodium salt, 75 to 324 micromol/kg, produced a net iron excretion that was nearly three times that observed after similar doses of sc DFO. In patients with transfusional iron overload, sc injections of HBED may provide a much needed alternative to the use of prolonged parenteral infusions of DFO. Note: After the publication of our previous paper (Blood, 91:1446, 1998) and the completion of the studies described here, it was discovered that the HBED obtained from Strem Chemical Co (Newburyport, MA) that was labeled and sold as a dihydrochloride dihydrate was in fact the monohydrochloride dihydrate. Therefore, the actual administered doses were 81, 162, or 324 micromol/kg; not 75, 150, or 300 micromol/kg as was previously reported. The new data have been recalculated accordingly, and the data from our earlier study, corrected where applicable, are shown in parentheses.     

The next generation of research: views of a sibling-psychiatrist-researcher

Ceruloplasmin purified from horse serum was rapidly reduced upon addition of increasing equivalents of ferrous iron, generating an electronically and conformationally distinct form. This form of ceruloplasmin was characterized by significant (80%) loss of EPR detectable type I and type II copper(II), complete loss of visible absorbance at 610 nm, as well as decreased hydrophobic surface area. The reduced form of ceruloplasmin slowly reduced molecular oxygen to complete its catalytic cycle. The presence of varied concentrations of apoferritin, but not apotransferrin, significantly enhanced the rate of ceruloplasmin oxidation. The magnitude of this stimulatory effect increased as the molar ratio of ceruloplasmin to apoferritin approached 1.0, shown previously to be the optimum ratio for loading iron into ferritin. The rate of ferrous iron oxidation by ceruloplasmin was significantly stimulated by the presence of apoferritin; however, apotransferrin had no effect. The length of time required for ceruloplasmin to oxidize all the iron and return to the native form of the enzyme was also affected by the concentration of iron. In addition, the rate of iron loading into ferritin was dependent upon ferrous iron concentration. These results provide evidence for the formation of a specific complex between the reduced form of ceruloplasmin and apoferritin and that reduction of ceruloplasmin by ferrous iron may be the signal for complex formation.