Radioactive Iron Research Articles

Validation of a method of treating menorrhagia by endometrial ablation.

Menstrual blood loss was measured by intravenous injection of radioactive iron (59Fe) and whole-body counting in 19 women complaining of menorrhagia due to dysfunctional bleeding. Fifteen patients were then treated by radiofrequency endometrial ablation, after which blood loss was re-measured. The majority of the patients were bleeding excessively before treatment (mean 281 (SD 156) ml per cycle averaged over two cycles, range 49-665 ml, n = 19). In the 15 treated patients, average blood loss was reduced to 52 (SD 39) ml per cycle and the mean reduction in blood loss was highly significant (P < 0.001). These quantitative data correlate well with the patients' subjective reports.

Rhizobial siderophore as an iron source for clover

Iron uptake by clover plants (Trifoliuin pratense L. cv. Hruszowska) was studied using radioactive ferric iron (55FeCl2). As shown by autoradiography of non‐infected plants, purified rhizobial siderophore isolated from Rhizobium leguminosarum by, trifolii, stimulated the uptake and shoot transport of iron. Addition of rhizobial siderophore into the growth medium of nodulated clover did not affect the iron transport to the shoots. In the absence of the rhizobial siderophore, clover infected by either nitrogen‐fixing (Nod− Fix+) or nonfixing (Nod+ Fix−) R. L. trifolii strains took up and transported into the shoots more iron than the non‐infected control plants. Nodulated clover reduced Fe(III) more efficiently than the non‐infected control plants.

Quantification of desferrioxamine and its iron chelating metabolites by high-performance liquid chromatography and simultaneous ultraviolet-visible/radioactive detection

An HPLC-based method for quantification of desferrioxamine (DFO) and its iron chelating metabolites in plasma has been developed. This assay overcomes stability problems associated with DFO by the addition of radioactive iron to convert unbound drug and metabolites to radio-iron-bound species. A dual detection system utilizing uv-vis absorption and radioactive (β-particle) detector was used to quantify total and radio-iron-bound species. The use of octadecyl silanol solid phase extraction cartridges permits concentration of samples and allows accurate quantification of drug and metabolites down to 0.1 nmol/ml.

Cadmium treatment and lead‐induced suppression of splenic erythropoiesis

Young adult female mice were injected with lead acetate (d 0). Following injection, determinations were made of the percentages of radioactive iron (59Fe) uptake into the hemoglobin of erythrocytes produced by spleen. Control 59Fe uptake percentage vacillated between 4.2 and 5.5 within the 7-d period of observation. On d 4 following lead treatment, splenic percentages were dramatically reduced below those of the saline-injected controls; by d 6 the splenic 59Fe uptake of lead-treated mice was comparable to that of controls. For rodents injected with cadmium chloride on 0, the 59Fe uptake values showed a statistically significant elevation by d 2, which was extended beyond that of the controls' d 4 value. For those animals receiving both lead and cadmium (d 0), the uptake percentages paralleled those of the controls throughout the 7-d period of observation. These data suggest that the inhibitory effect of lead on erythropoiesis of the spleen is blocked by a concurrent cadmium treatment. Results are interpreted in regard to a possible vulnerable target and competition for the target by lead and cadmium.

Transferrin Receptor Expression and Iron Uptake in the Injured and Regenerating Rat Sciatic Nerve.

Iron-saturated transferrin is a ubiquitous growth factor that plays a critical role in cellular iron uptake, growth and proliferation. Here we have studied the expression and distribution of transferrin receptors and iron uptake following injury of the rat sciatic nerve. Axotomy led to a massive but transient increase (days 2 - 9, maximum day 4) in [125I]transferrin binding at the site of the injury and in the distal, denervated part of the crushed or resected sciatic nerve, shortly preceding the time course of cellular proliferation (Friede and Johnstone, Acta Neuropathol, 7, 218 - 231, 1967; Jurecka et al., Acta Neuropathol, 32, 299 - 312, 1975). An additional, transient increase in specific binding was observed during reinnervation after reconnection of the resected sciatic nerve. Immunocytochemistry using the Ox-26 monoclonal antibody revealed strong and simultaneous expression of the transferrin receptor protein on two different cell types: on a subpopulation of blood-borne macrophages invading the injured peripheral nerve and on Schwann cells reacting to denervation and reinnervation. In addition, studies using intravenously injected radioactive iron (59Fe3+) showed a massive increase in endoneural iron uptake confined to the lesion site and to the distal part of the axotomised sciatic nerve, parallel to the time course of reactive transferrin receptor expression. Since iron is an essential cofactor of a number of key enzymes needed in energy metabolism and DNA synthesis, these data suggest that the induction of transferrin receptor expression may play an important role in the regulation of cellular growth and proliferation during peripheral nerve regeneration.

Investigation of the aetiology of haemosiderosis in the starling(Sturnus vulgaris)

Birds with haemosiderosis absorbed up to 10% of an orally administered test dose of radioactive iron, even when the hepatic iron concentrations were markedly increased. In contrast, rats which were orally loaded with iron to produce a comparable hepatic iron concentration absorbed only ' about 1% of administered (59)Fe. It is concluded that the absorption of iron at the intestinal mucosa in avian species is not as tightly regulated as that in some mammals.

Effects of naturally occurring iron-carriers on continuously cultivated Escherichia coli

Previous work has shown that when fulvic acid, which occurs naturally in Birmingham tap-water, is extracted and added, along with iron, to continuous cultivation media, the mean cell volume increases with agitation rate at constant dilution rate. The organic material appears to act as an iron-carrier. These effects are examined in more detail in the present paper in which the effects produced by tap-water are compared with those produced by peat extracts. The fulvic acid extracted from Birmingham tap-water was compared with fulvic acid extracted from peat. Both materials produced volume increases in continuously cultivated cells in the presence of iron, and it was concluded that both were effective as iron-carriers. Direct aqueous extracts of peat also proved very active in this respect. Use of radioactive iron as a tracer showed that the amount of iron associated with the continuously cultivated microbial cells increased with increase in agitation rate in both tap-water based medium and in distilled water based medium containing peat extracts, whereas no such increase was observed in the case of distilled water based control medium. It is concluded that the distribution of such natural iron-carriers of this type is quite widespread, and that they are involved in mechanisms of iron uptake and extraction for a wide range of living organisms.

Reduced hepatic iron uptake from rat aglycotransferrin

Rat aglycotransferrin (rAgTf) was produced from the disialosyl diantennary fraction of rat transferrin (rTf) by treatment with peptide: N-glycosidase F. Following removal of the enzyme by gel filtration and isolation of the deglycosylated protein by lectin chromatography, rAgTf was compared to rTf both in vitro and in vivo. No significant differences were found between the two proteins with respect to affinity for iron and kinetics of Fe release from the N-lobe and C-lobe. The fluorescence emission spectrum of apo-rTf was red-shifted by approximately 3 nm relative to diferric rTf; however, no spectral difference was detected between rTf and rAgTf when the analogous forms (apo or diferric) were compared. Plasma clearance of radioactive iron administered to rats as either rTf or rAgTf was comparable. Reticulocytes took up iron from rAgTf slightly faster than from rTf. In contrast, Fe acquisition by the liver from rAgTf was significantly reduced relative to rTf. This finding contrasts sharply with earlier observations with asialotransferrin (rAsTf) and provides a basis for discounting charge loss as the mechanism of enhanced hepatic Fe uptake from rAsTf. It is suggested that the glycan complement of rTf, while unimportant for interaction of the protein with specific receptors, probably plays a role in the interaction with low-affinity hepatic binding sites.

Acute effects of T-2 toxin on radioactive iron incorporation into circulating erythrocytes in mice

The 24-h and 72-h incorporation of 59Fe into circulating erythrocytes in mice were strongly inhibited by a single subcutaneous dose of T-2 toxin given 1 h before the radioisotope. The system is extremely sensitive, since a significant effect was detected with T-2 toxin doses as low as 0.30 mg/kg, which is about one-tenth of the LD 50 in the BALB/c strain used for the present study. In the treated animals no initial changes were observed in the blood 59Fe levels or in the rate of radioisotope clearance from plasma, indicating that the toxin does not interfere with iron absorption or transport. It is concluded that the inhibition observed reflects the damage produced by this toxin on reticulocytes and/or erythroblasts, and therefore this method could be of value as a very sensitive means of studying the risk of erythropoietic injury produced by dietary exposure to trichothecene mycotoxins.

Cir and Fiu proteins in the outer membrane of Escherichia coli catalyze transport of monomeric catechols: study with beta-lactam antibiotics containing catechol and analogous groups

Recently, beta-lactam agents containing iron-chelating moieties, such as E0702, which contains catechol, and pirazmonam and U-78,608, which contain 3-hydroxypyridone, have been developed. By determining the susceptibility to these agents of Escherichia coli mutants lacking various iron-repressible outer membrane proteins, we showed that two of these proteins with hitherto unknown functions, Fiu and Cir, were apparently involved in the transport of monomeric catechol and its analogs. These results confirm the conclusion of Curtis and co-workers, which was obtained by using a different set of catechol-containing antibiotics (N. A. C. Curtis, R. L. Eisenstadt, S. J. East, R. J. Cornford, L. A. Walker, and A. J. White, Antimicrob. Agents Chemother. 32:1879-1886, 1988). E0702 was shown to enhance the uptake of radioactive ferric iron into wild-type cells but not into cir fiu double mutants. By combining the influx of E0702 with its hydrolysis by a periplasmic beta-lactamase, we showed that the wild-type cells transported unliganded E0702 at a rate comparable to or even higher than the rate of transport of the E0702-Fe3+ complex. We postulate that the main function of Cir and Fiu may be to recapture the hydrolytic products of enterobactin, such as 2,3-dihydroxybenzoic acid and 2,3-dihydroxybenzoylserine.

A comparison of hematopoiesis in normal and splenectomized mice treated with granulocyte colony-stimulating factor

Recombinant human granulocyte colony-stimulating factor (rhG-CSF) induces leukocytosis in vivo in both intact and splenectomized mice. Full dose response data showed a plateau in this effect at doses over 500 micrograms rhG-CSF/kg body weight/d in intact mice. The effect is magnified in splenectomized mice, where leukocyte numbers reach 100 x 10(6) mL after 4 days' treatment at 250 micrograms/kg/d. Further hematopoietic precursor populations are also affected in both marrow and the spleen; in general, marrow parameters were depressed, while splenic populations were enlarged. In splenectomized mice, both blood-borne stem cells were enhanced, and foci of extramedullary hematopoiesis were enlarged in addition to the effects seen in intact mice. In the marrow of splenectomized and intact mice treated with a high dose of G-CSF, erythroid suppression in the marrow was confirmed with radioactive iron. Our studies confirm and extend previous work on the mode of action of G-CSF, and indicate that side effects of high dose G-CSF therapy might include erythroid suppression in the bone marrow.

A Comparison of Hematopoiesis in Normal and Splenectomized Mice Treated With Granulocyte Colony-Stimulating Factor

Abstract Recombinant human granulocyte colony-stimulating factor (rhG-CSF) induces leukocytosis in vivo in both intact and splenectomized mice. Full dose response data showed a plateau in this effect at doses over 500 micrograms rhG-CSF/kg body weight/d in intact mice. The effect is magnified in splenectomized mice, where leukocyte numbers reach 100 x 10(6) mL after 4 days' treatment at 250 micrograms/kg/d. Further hematopoietic precursor populations are also affected in both marrow and the spleen; in general, marrow parameters were depressed, while splenic populations were enlarged. In splenectomized mice, both blood- borne stem cells were enhanced, and foci of extramedullary hematopoiesis were enlarged in addition to the effects seen in intact mice. In the marrow of splenectomized and intact mice treated with a high dose of G-CSF, erythroid suppression in the marrow was confirmed with radioactive iron. Our studies confirm and extend previous work on the mode of action of G-CSF, and indicate that side effects of high dose G-CSF therapy might include erythroid suppression in the bone marrow.

Failure to label baboon milk intrinsically with iron.

The widely held belief that 50% of the iron in human milk is absorbed is based on studies that have used an extrinsic radioactive iron tag. To determine the validity of an extrinsic tag, it is necessary to label the milk intrinsically with one isotope and to compare absorption of this isotope with absorption of another isotope added as the extrinsic tag. We chose the baboon as a model and infused 59Fe intravenously. In each of three attempts we failed to label the milk intrinsically.

Failure to Label Baboon Milk Intrinsically with Iron

The widely held belief that 50% of the iron in human milk is absorbed is based on studies that have used an extrinsic radioactive iron tag. To determine the validity of an extrinsic tag, it is necessary to label the milk intrinsically with one isotope and to compare absorption of this isotope with absorption of another isotope added as the extrinsic tag. We chose the baboon as a model and infused 59Fe intravenously. In each of three attempts we failed to label the milk intrinsically.

Analysis of density-fractionated rat red blood cells of different ages by partitioning in two-polymer aqueous phase systems

Rats were injected with radioactive iron and bled at different times thereafter. This gave rise to cell populations in which the bulk of labeled cells corresponded in age to the time elapsed between injection and bleeding. Such cell populations were centrifuged and the 7–10% least dense and the 7–10% most dense cells subjected to counter-current distribution in a charge-sensitive two-polymer aqueous phase system which fractionates cells on the basis of surface properties. While it is known that there is a tendency for cell density to increase with cell age and that rat red cells of different ages have characteristic partition ratios, we were able, by applying partitioning analysis to density-separated cells, to gather data not obtainable by use of either method alone. These include: (1) the demonstration of two very young cell populations having different surface properties in the most dense fraction; (2) the finding that in the least dense layer labeled mature erythrocytes (at least those labeled 18 days and beyond) and unlabeled red cells have overlapping distribution curves which indicate that the label in the top fraction can represent young or early middle-aged but never old cells; and (3) the presence, as judged by surface properties, of some middled-aged red cells, in addition to old cells, in the most dense cell layer.

Effect of chronic ethanol administration on iron metabolism in the rat.

This study shows that the ingestion of ethanol provokes alterations in iron metabolism which may lead to iron overload. Impaired release of reticuloendothelial iron was shown by a decrease of the maximum red blood cell utilization when radioactive iron was supplied as colloidal iron. An impairment in the erythropoietic activity of ethanol-treated animals was also observed, as can be seen from the reduced plasma iron turnover and red blood cell utilization within 24 h of iron administration. A rise in marrow transit time was also observed. In ethanol-treated rats there was an increase in the amount of iron retained both in the liver and the spleen. This was observed in both sexes and also in the offspring from ethanol-treated mothers.

Mitochondrial iron loss from leukemia cells injured by macrophages. A possible mechanism for electron transport chain defects.

Activated macrophages inhibit replication of murine lymphoblastic leukemia L1210 cells without lysis. This inhibition of replication is associated with abnormalities of mitochondrial electron transport at the level of NADH dehydrogenase (NADH-DH) and succinate dehydrogenase (SDH). The mechanism of inhibition is unknown, although it has been demonstrated that as NADH-DH and SDH activity is lost, iron is released from cells. Because both NADH-DH and SDH contain numerous iron-sulfur clusters, damage to these structures may be one result of injury by activated macrophages. L1210 cells were labeled with 55Fe and co-cultivated with activated murine peritoneal macrophages (injured L1210 cells). At 48 h, injured L1210 cells had released 83 +/- 8% (mean +/- SEM of 55Fe activity into the media, compared with 25 +/- 4% release from control and 37 +/- 7% from nondividing mitomycin C-treated control cells. All cells were greater than 90% viable. These differences were also reflected in the iron content of the cells. Mitochondria were then separated by centrifugation after cell disruption and 55Fe activity was found to be similarly decreased in both mitochondrial and nonmitochondrial fractions of injured L1210 cells. To further characterize the changes in mitochondrial iron content, mitochondrial proteins from injured and control L1210 cells were separated by IEF and 55Fe activity of gel slices was determined. There was selective loss of 55Fe activity in the area of the gel corresponding to SDH and NADH-DH, suggesting that iron loss from iron-sulfur clusters may occur in L1210 cells injured by activated macrophages. Iron uptake into L1210 cells after removal from macrophages showed a rapid large influx of radioactive iron. L1210 cells in contact with macrophages appear to develop an iron-depleted state, which is dependent on the continued presence of macrophages.

Characteristics of iron(III) uptake by isolated fragments of rat small intestine in the presence of the hydroxypyrones, maltol and ethyl maltol

Accumulation of radioactive iron ( 59Fe) into isolated fragments of rat small intestine in the presence of two hydroxypyrones, maltol and ethyl maltol, was compared with that in the presence of another chelator of iron(III), nitrilotriacetic acid (NTA). The characteristics of uptake were similar with all three ligands. Between 10 −6 and 10 −4 M, iron uptake showed saturable kinetics. The uptake was partially inhibited by metabolic inhibitors. Above 10 −4 M a non-saturable uptake, unaffected by metabolic inhibitors became evident in the presence of the pyrones. The distribution of 59Fe after uptake was determined by gel filtration. At low iron concentrations (10 −6M), 35–40% of absorbed iron was associated with proteins of molecular weights similar to those of ferritin and transferrin. At high concentrations (10 −3M), the majority of 59Fe was found in a low molecular weight fraction. At each concentration, a small amount of 59Fe was bound to a membrane fraction. 5% Polyethylene glycol, which reduces glycocalyx viscosity enhanced uptake at low iron concentrations (10 −6M) but did not affect the non-saturable diffusion seen at higher concentrations (10 −3M). The iron(II) chelator, bathophenanthroline sulphonate (10 −3M), decreased uptake at low iron concentrations but did not affect the non-saturable uptake. It is suggested that conversion of iron(III) to iron(II) may take place at the mucosal cell surface before uptake via the saturable system. Apparent K m values for iron uptake via the saturable system were higher in the presence of maltol and ethyl maltol than in the presence of NTA, presumably since the iron binds more avidly to the hydroxypyrones and so is less readily donated. Excess ligand, either pyrone or NTA, reduced the rate at which 59Fe was donated to the uptake system. The V max value for uptake from the pyrones was greater than from NTA. It is concluded that maltol, ethyl maltol and NTA can hold iron(III) in solution and donate it to an endogenous uptake system. But, the hydroxypyrones may be more suitable ligands for the oral administration of iron since, when complexed with iron, they lack the toxic effects associated with iron(III)-NTA and with iron(II) preparations.

The effect of soybean phosphate and phytate content on iron bioavailability

To study the inhibitory effects of soybeans on nonheme iron absorption soybeans were grown in hydroponic culture and labelled intrinsically with radioactive iron. Culture conditions were adjusted to produce seeds with high, intermediate, and low phosphate and phytate content. No effect of phosphate/phytate content on iron absorption was observed in radioiron absorption studies in rats. Absorption tests in human volunteers comparing hydroponically cultured low phosphate/phytate beans with moderate phosphate and phytate beans also failed to demonstrate a significant inhibitory effect of the phosphate or phytate. These observations do not support the hypothesis that phytate is the factor in soybeans and soy protein products that reduces iron bioavailability.

Subcellular distribution of recently absorbed iron and of transferrin in the mouse duodenal mucosa

A successful method for the analytical subcellular fractionation of mouse duodenal mucosa organelles was established. 59Fe(III)-nitrilotriacetate (pH 7.2) was injected into tied-off duodenal segments in vivo and, after 2–20 min, mucosal homogenates were subjected to subcellular fractionation. Radioactivity was recovered in the cytosolic fractions and in the gradient at a density of 1.18–1.20 g/ml. Enhanced iron absorption was achieved by placing the animals in a hypobaric chamber for 3 days. These animals had a higher proportion of particulate 59Fe compared to controls. Homogenisation in sucrose medium containing the selective plasma membrane perturbant digitonin shifted the particulate iron fraction to a higher density region of the gradient indicating a localisation of the iron binding site to the plasma membrane region of the mucosal cells. No significant radioactive iron was observed in the brush-border region of the gradient. Transferrin immunoreactivity was found only in the cytosolic region of the gradient and was not associated with any organelle.