The utilization of senescent red cell and hemolysate iron for erythropoiesis.

We report experiments to determine the availability for new hemoglobin production of radioiron from nonviable red cells at various times after deposition in the reticulo-endothelial system and to determine the relative availability of radioiron derived from hemolysates versus that derived from nonviable red cells. When heated nonviable red cells labeled with 59Fe are injected into polycythemic mice the iron is deposited in the reticulo-endothelial system, and less than 1% of it is reutilized for hemoglobin synthesis. If the polycythemic mice are given nonviable red cells 48 hours after exposure to hypoxia, when hemoglobin synthesis is maximal, 25% of the iron is reutilized. When the cells are given 36 hr after exposure to hypoxia, iron reutilization declines to 16%, and when exposure to hypoxia is further delayed, reutilization of the iron falls to a plateau level of 11%. Radioiron from hemolysates, primarily deposited in parenchymal cells of the liver, is less available for new hemoglobin synthesis than is radioiron from nonviable red cells, which is primarily deposited in Kupffer cells of the liver. When transferrin-bound iron is given to polycythemic mice, this iron is also deposited in parenchymal cells of the liver and is also less available for new hemoglobin synthesis. Thus, in relation to an erythropoietic stimulus, the site and time of deposition of iron influence its accessibility for erythropoiesis.     

Biochemistry of nonheme iron in man. II. Absorption of iron

The currently accepted concept of iron absorption proposes first the entry of iron into the intestinal mucosal cell through the brush border membrane. It is a relatively slow process. In the cell, the iron may be transferred to plasma or become sequestered by ferritin. The latter becomes unavailable for transfer to plasma and is exfoliated and excreted. In iron deficiency and idiopathic hemochromatosis, the rate of iron uptake into the intestinal mucosal cell is increased and entry into ferritin is decreased, whereas the rate of transfer to plasma remains constant. The reverse occurs in case of secondary iron overload. It is currently accepted that a transferrin, whose levels increase in iron deficiency, enters the intestinal lumen from the liver via bile, where it may sequester iron and bring it into the cells by the process of endocytosis. Iron presented as inorganic ferric or ferrous salts may also be absorbed, though the more soluble ferrous salts are adsorbed much more rapidly. Heme iron is absorbed very effectively, though it is not subject to regulation by the individual's iron status to the same extent as is inorganic iron absorption. Brush border membranes apparently contain saturable iron receptors for inorganic iron, but whether or not the absorption process requires energy is an open question. Absorption of iron may also be affected by its availability; different food components affect iron absorbability to a different extent.     

The effect of short-term exposure to low-iron diets on the mucosal processing of ionic iron

Short-term alterations in the amount of iron in the diets of rats caused substantial differences in the distribution of a test dose of radioiron between mucosal transferrin and mucosal ferritin, and also caused a change in the relative amounts of these two proteins in mucosal tissue without resulting in any detectable change in liver iron stores. These differences correlated with changes in the retention of radioiron by the intestinal mucosa and the transport of radioiron to the blood stream. These studies emphasize the importance of local changes in the intestinal mucosa in the regulation of dietary iron absorption.     

Copper deficiency increases iron absorption in the rat

Release of iron from enterocytes and hepatocytes is thought to require the copper-dependent ferroxidase activity of hephaestin (Hp) and ceruloplasmin (Cp), respectively. In swine, copper deficiency (CD) impairs iron absorption, but whether this occurs in rats is unclear. By feeding a diet deficient in copper, CD was produced, as evidenced by the loss of copper-dependent plasma ferroxidase I activity, and in enterocytes, CD reduced copper levels and copper-dependent oxidase activity. Hematocrit was reduced, and liver iron was doubled. CD reduced duodenal mucosal iron and ferritin, whereas CD increased iron absorption. Duodenal mucosal DMT1-IRE and ferroportin1 expression remained constant with CD. When absorption in CD rats was compared with that seen normally and in iron-deficient anemic animals, strong correlations were found among mucosal iron, ferritin, and iron absorption, suggesting that the level of iron absorption was appropriate given that the erythroid and stores stimulators of iron absorption are opposed in CD. Because CD reduced the activity of Cp, as evidenced by copper-dependent plasma ferroxidase I activity and hepatocyte iron accumulation, but iron absorption increased, it is unlikely that the ferroxidase activity of Hp is important and suggests another function for this protein in the export of iron from the enterocyte during iron absorption. Also, the copper-dependent ferroxidase activity of Cp does not appear important for iron efflux from macrophages, because Kupffer cells of the liver and nonheme iron levels of the spleen were normal during copper deficiency, suggesting another role for Cp in these cells.     

The valency state of absorbed iron appearing in the portal blood and ceruloplasmin substitution

Summary (1) Attempts to determine the redox-state of the absorbed iron, which appeared in the portal blood when the free iron-binding capacity was previously saturated, indicate that about 30–90% of this iron was in the ferrous state. This effect was particularly prominent after luminal administration of ferrous iron, but was also seen when iron was given in the ferric state. (2) Total iron absorption is significantly higher in ceruloplasmin-substituted copper-deficient animals as compared to copper-deficient controls. (3) The appearance rate of absorbed iron in the portal blood of copper-deficient animals increased several times immediately after the intravenous infusion of ceruloplasmin. (5) The distribution of absorbed iron was changed due to the ceruloplasmin substitution: it was increased in the reticulocytes (+66%), plasma (+400%) and the body (+ 112%), whereas in the liver it was decreased by about 78%. (5) In iron-deficient rats intravenously injected ceruloplasmin did not increase iron absorption. (6) The conclusion was drawn that, as for the entrance into the mucosa from the luminal side, also for the release at the contraluminal side into the portal blood, the ferrous state of iron is favoured and that ceruloplasmin accelerates the release into the portal blood by catalyzing the oxidation of ferrous iron due to its high Fe(II):oxygen oxidoreductase (EC 1.16.3.1) activity.     

Iron absorption in the iron-deficient rat

Iron absorption in the iron-deficient rat was compared with that in the normal rat to better understand the regulation of this dynamic process. It was found that: Iron uptake by the iron-deficient intestinal mucosa was prolonged as a result of slower gastric release, particularly when larger doses of iron were employed. The increased mucosal uptake of ionized iron was not the result of increased adsorption, but instead appeared related to a metabolically active uptake process, whereas the increased mucosal uptake of transferrin iron was associated with increased numbers of mucosal cell membrane transferrin receptors. Mucosal ferritin acted as an iron storage protein, but its iron uptake did not explain the lower iron absorption in the normal rat. Iron loading the mucosal cell (by presenting a large iron dose to the intestinal lumen) decreased absorption for 3 to 4 days. Iron loading of the mucosal cell from circulating plasma transferrin was proportionate to the plasma iron concentration. Mucosal iron content was the composite of iron loading from the lumen and loading from plasma transferrin versus release of iron into the body. These studies imply that an enhanced uptake-throughout mechanism causes the increased iron absorption in the iron-deficient rat. Results were consistent with the existence of a regulating mechanism for iron absorption that responds to change in mucosal cell iron, which is best reflected by mucosal ferritin.     

The effects of cytoskeletal inhibitors on intestinal iron absorption in the rat

An established and validated method using loops of intestine in vivo in rats was used to study the effects of cytoskeletal inhibitors on iron absorption. Radioactive iron instilled into the loop of intestine pretreated with test substance was monitored in the blood and, after death, ferritin loading with radioactive iron was measured on density gradients of mucosal cell homogenates and absorbed iron in the carcass was determined. Colchicine, vincristine and cytochalasin B all caused dose- and time-dependent inhibition of iron absorption, and the effects of cytochalasin B were reversible within 1 h. It is not known which cellular component is the vehicle for the transcellular movement of iron from the intestinal lumen onto plasma transferrin; however, this study showed that the uptake of iron by ferritin in an iron-absorbing loop of intestine paralleled the actual absorption of iron into the carcass. This phenomenon did not occur in non-iron-absorbing intestinal and was inhibited by the action of the cytoskeletal inhibitors in the iron-absorbing region. Previously we had shown that iron uptake into cells and onto cellular transferrin was virtually the same throughout the small intestine, irrespective of the iron-absorbing capacity of the region. The results of this study therefore suggest that iron absorption depends on an intact cytoskeletal system and that ferritin in the iron-absorbing cell is able to load from the pool of iron committed to transcellular movement onto plasma transferrin.     

Distribution of55Fe in juvenile adult lampreys,Petromyzon marinus L., following oral intubation of the isotope

Summary The capacity of a sanguivorous lamprey,Petromyzon marinus L., to deal with ingested iron was studied over time using autoradiography and scintillation counting of solubilized tissue samples after intubation of the oesophagus with a single dose of⁵⁵ferrous citrate. A highly efficient mechanism for absorption in the anterior intestine was recognized with 17% of the intubated radioactivity absorbed into the body after only 5 min, 66% by 3 h, and almost 80% by 21 h. Iron concentration in the epithelial cells of the anterior intestine may be a factor in restricting iron absorption during spontaneous feeding. A decline in total body radioactivity over the 15 days following iron intubation probably results from transport of the metal in the blood and release of radioiron from the mucous cells of the posterior intestine. The kidneys appear to play a smaller but still significant role in iron loss. Gradual increases in radioiron concentration (cpm g^–1 wet weight) and percent of total body radioactivity occur in the liver (2 to 26%), carcass (14 to 37%), and integument (4 to 12%) during the course of the experiment, indicating that these are the chief sites of iron storage during times of metal excess. However, eventually integument may also be a site of iron excretion. Significant fluctuations in radioiron concentration (cpm ml^–1) in whole blood during the 15 day period can be correlated with transport of the metal to sites of storage and excretion, and maybe with incorporation into haemoglobin and with erythropoietic activity. Feeding adult lampreys represent a valuable system, with both general and unique characters, for studying iron metabolism in vertebrates.     

The induction of ferritin synthesis in circulating larval red blood cells.

The circulating red blood cells formed in bullfrog larvae, chicken embryos, and mouse embryos contain large amounts of ferritin and storage iron in excess of the need for hemoglobin. In contrast, the circulating red cells of adult animals contain little ferritin. Ferritin synthesis and iron storage are coordinated with differentiation and hemoglobin synthesis in the red cells of adults. In order to test the hypothesis that ferritin synthesis could be controlled independently of hemoglobin synthesis and differentiation in the red cells formed early in life, bullfrog larvae were injected with iron to determine if ferritin synthesis was increased in the circulating red cells. Within 17 h after the injection of iron, the synthesis of ferritin, assayed as the incorporation of [14C]leucine by cell suspensions prepared from circulating red cells, was increased from 2.9 to 10.2% of the total protein, and the specific activity of the ferritin synthesized increased from 1100 to 3000 cpm/A280. There was no change in the hematocrit of the animals nor in the specific activity of hemoglobin synthesized by suspensions of red cells (average, 720 cpm/A280). The results suggest that in mature, larval red cells, ferritin synthesis can be controlled by changes in the extracellular environment. The results also indicate that ferritin synthesis can be controlled independently of hemoglobin synthesis with which it is coordinated during erythroid differentiation in adult animals.     

Effects of acute iron overload on Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss)

Distribution of radioiron to various tissues after intraperitoneal injections was examined in Atlantic salmon and rainbow trout. Liver and spleen were found to be the major iron storage tissues. Injections of 1 or 5 mg iron as ferric ammonium citrate led to a fall in hemoglobin levels in both species after 2 d. Hemoglobin levels returned to normal levels in rainbow trout after 8 d, but Atlantic salmon had not recovered, and Hb levels fell below 3 g/100 mL. In both species, the fall in Hb was associated with a raise in iron levels in spleen and liver, suggesting damage to erythrocytes. Atlantic salmon liver ferritin showed a two- to threefold increase, while rainbow trout showed a sixfold increase, and a more rapid response. The toxic effect of iron in fish appears to be different from the effect in other vertebrates.     

Factors involved in the regulation of iron transport through reticuloendothelial cells

The effects of various maneuvers on the handling of 59Fe-labeled heat-damaged red cells (59Fe HDRC) by the reticuloendothelial system were studied in rats. Raising the saturation of transferrin with oral carbonyl iron had little effect on splenic release of 59Fe but markedly inhibited hepatic release. Splenic 59Fe release was, however, inhibited by the prior administration of unlabeled HDRC or by the combination of carbonyl iron and unlabeled HDRC. When carbonyl iron was administered with unlabeled free hemoglobin, the pattern of 59Fe distribution was the same as that observed when carbonyl iron was given alone. 59Fe ferritin was identified in the serum after the administration of 59Fe HDRC but the size of the fraction was not affected by raising the saturation of transferrin. Sizing column analyses of tissue extracts from the spleen at various times after the administration of 59Fe HDRC revealed a progressive shift from hemoglobin to ferritin, with only small amounts present in a small molecular weight fraction. The small molecular weight fraction was greater in hepatic extracts, with the difference being marked in animals that had received prior carbonyl iron. The increased hepatic retention of 59Fe associated with a raised saturation of transferrin was reduced by a hydrophobic ferrous chelator (2,2'-bipyridine), a hydrophilic ferric chelator (desferrioxamine), and an extracellular hydrophilic ferric chelator (diethylene-triaminepentacetic acid). Transmembrane iron transport did not seem to be a rate-limiting factor in iron release, since no differences in 59Fe membrane fractions were noted in the different experimental settings. These findings are consistent with a model in which RE cells release iron from catabolized red cells at a relatively constant rate. When the saturation of transferrin is raised, a significant proportion of the iron is transported from the spleen to the liver either in small molecular weight complexes or in ferritin. Although a saturated transferrin had no effect on the release of iron from reticuloendothelial cells, prior loading with HDRC conditions them to release less iron.     

Structure and expression of genes involved in transport and storage of iron in red-blooded and hemoglobin-less antarctic notothenioids

Antarctic notothenioids are characterized by a drastic reduction of the hemoglobin content, a condition that reaches its extreme in icefish that, following a gene deletion event, are completely devoid of hemoglobin. To answer the question on what type of adaptive changes occurred in icefish to prevent accumulation of potentially dangerous ferrous iron, we investigated the genes of four proteins known to play a key role in iron metabolism. For this purpose, we cloned and sequenced the cDNAs encoding ceruloplasmin, transferrin, ferritin and divalent metal transporter 1. While the inferred amino acid sequences of transferrin from different Antarctic fish species showed a high level of similarity with the homologous proteins from other species, ceruloplasmin sequence featured amino acid substitutions affecting a copper binding site. Another peculiarity was the presence in subunit H of the icefish ferritin of the two sets of sites involved in iron oxidation and iron mineralization, which in mammals are located on two distinct ferritin subunits. Significant differences in the expression levels of the four genes were found between hemoglobinless and red-blooded notothenioids. An increased expression of ceruloplasmin mRNA in icefish was interpreted as a compensatory mechanism to prevent accumulation of ferrous iron in hemoglobinless fish. In icefish, the amounts of ferritin H-chain mRNA measured in liver, blood and head kidney were lower than in the same organs of the red-blooded fish. In the spleen of both fishes, the expression levels of ferritin H-chain were significantly lower than in the spleen of a "pink-blooded" notothenioid with an intermediate hemoglobin content. Finally, the amount of divalent metal transporter mRNA measured in the head-kidney was lower in the icefish than in the same organ of its red-blooded counterpart. These results indicate that the loss of hemoglobin in icefish is accompanied by remodulation of the iron metabolism.     

The effect of ferrous sulfate and pH on L-dopa absorption

Ferrous sulfate decreases L-dopa bioavailability in humans probably as a result of binding of L-dopa by iron in the gastrointestinal tract. This study was conducted to determine if iron by binding L-dopa decreases L-dopa absorption and to investigate the effect of different pH buffers on intestinal absorption of L-dopa in the presence and absence of ferrous sulfate. A rat model developed to examine drug absorption was used. Control animals had buffered [14C]L-dopa solutions injected into two in vivo closed segments of intestine; a 5-cm duodenal and a 5-cm proximal jejunal segment. These studies were conducted using solutions buffered at pH 5.5, 6.5, 7.5, and 8.5. An identical procedure was followed for experimental animals except ferrous sulfate was injected with the buffered L-dopa solutions. Ferrous sulfate resulted in a reduction in L-dopa absorption in the buffers at all pHs in both the duodenum and jejunum. The average reduction in L-dopa absorption in the presence of iron was 22.6% in the duodenum and 23.9% in the jejunum. There was a tendency for ferrous sulfate to cause a greater reduction in L-dopa absorption as the buffer pH increased. There was also a decrease in L-dopa absorption in the higher pH buffers in the absence of iron. Despite this latter result, in the jejunum there was an increase in the percent reduction in L-dopa absorption associated with ferrous sulfate as pH increased. Although this tendency was not as consistent in the duodenum as the jejunum, the combined results are compatible with the chemical model of increased L-dopa--iron binding as pH increases.     

HEMOGLOBIN ABSORPTION BY THE CELLS OF THE PROXIMAL CONVOLUTED TUBULE IN MOUSE KIDNEY

The formation of protein absorption droplets in the cells of the proximal convolution was studied in mouse kidney. Ox hemoglobin was administered intraperitoneally and kidney specimens were collected at intervals of 30 minutes to 4 days after injection. In the lumen of the nephron, hemoglobin was concentrated to an opaque mass whose relations with the brush border and the epithelium could be easily followed. It was found that hemoglobin passes through the brush border in between the microvilli, enters the channels of tubular invaginations at the bases of the brush border, and is transported in bulk into vacuoles in the intermediate cell zone. These vacuoles increase in size and are transformed through further concentration into dense absorption droplets. Using the opaque hemoglobin content of the nephron as a tracer, functional continuity of the system of the tubular invaginations with the lumen on one side and the vacuoles on the other was demonstrated. Mitochondria lie closely apposed to vacuoles and droplets, but are not primarily involved in droplet formation. 15 hours after injection and later, ferritin and systems of layered membranes become visible in the droplets as their density decreases. These membranes are interpreted as lipoprotein membranes; similar membranes are found in the lumen of the tubuli. It is suggested that phospholipids enter into the vacuoles together with hemoglobin from the tubular lumen and form membrane systems of lipoproteins in the droplets. At 3 to 4 days the droplets contain aggregates of ferritin, and the iron reaction becomes positive in the tubule cells. No significant changes were found in the Golgi apparatus or in the microbodies during hemoglobin absorption. At all time points investigated, the terminal bars seal the intercellular spaces against penetration by hemoglobin in the proximal and distal convolutions and in the collecting ducts.     

Effects of fasting and/or oxidizing and reducing agents on absorption of neptunium from the gastrointestinal tract of mice and adult or neonatal rats

Neptunium-237(V) nitrate was administered by gavage to groups of fed or fasted adult and 5-day-old rats. Some groups also received the oxidants quinhydrone or ferric iron, and others received the reducing agent ferrous iron. Adult mice received ferric or ferrous iron and 235Np. When the adult rats were killed at 7 days after gavage, measurements showed that, compared with rats that were fed, a 24-hr fast caused a fivefold increase in 237Np absorption and retention. Both quinhydrone and ferric iron caused an even greater increase in absorption in both fed and fasted rats. Ferrous iron, on the other hand, decreased absorption in fasted rats to values lower than those obtained in fed rats. Similar results were obtained in mice treated with 235Np and either ferric or ferrous iron. The highest absorption obtained after gavage of ferric iron to fasted rats and mice was about two orders of magnitude higher than the value obtained in animals that were fed before gavage. The effects of ferric and ferrous iron on neptunium absorption by neonatal rats were similar to their effects on adult animals but of lesser magnitude. These results are consistent with the hypothesis that Np(V), when given in small mass quantities to fed animals, is reduced in the gastrointestinal tract to Np(IV), which is less well absorbed than Np(V).     

Ferrous iron transport in Streptococcus mutans.

Radioiron uptake from 59FeCl3 by Streptococcus mutans OMZ176 was increased by anaerobiosis, sodium ascorbate, and phenazine methosulfate (PMS), although there was a 10-min lag before PMS stimulation was evident. The reductant ascorbate may have provided ferrous iron. The PMS was reduced by the cells, and the reduced PMS then may have generated ferrous iron for transport; reduced PMS also may have depleted dissolved oxygen. We conclude that S. mutans transports only ferrous iron, utilizing reductants furnished by glucose metabolism to reduce iron prior to its uptake.     

Identification and characterization of the iron compounds in bone marrow by means of Mössbauer spectrometry

In order to determine and to demonstrate the cellular iron molecular states in hematopoietic bone marrow, direct investigations were performed by means of different and complementary spectroscopic techniques: optical absorption, electron spin resonance and Mössbauer spectrometry. In fact, the latter appears to have been the most informative. In addition to the hemoglobin forms, five- and six-coordination ligand protoporphyrins IX (monomeric and polymeric stacking, respectively) were observed. A small amount of non-hemic high-spin iron III storage component (ferritin) was measured. No diferric transferrin was detected. A ferrous compound was also observed and attributed to the mitochondrial iron pool.     

Rapid Induction of Ferritin in Laboratory Animals Prior to Its Isolation

Oral ingestion, by guinea pigs, of concentrated solutions of soluble hydroxy-iron(III) polymers rapidly induces a 7-fold increase of liver ferritin. Young guinea pigs imbibing 0.1 M iron for 2 weeks produced a significantly greater amount of ferritin than animals injected with an iron-dextran complex at 1.0 mmole Fe/kg body weight. Conventional methods for the isolation of tissue ferritin are more efficient and provide higher yields from such iron-replete animals. Dose/response curves are presented for the mouse to illustrate the kinetics of liver iron assimilation at various levels of oral iron supplementation.     

Iron homeostasis is dysregulated,but the iron-hepcidin axis is functional,in chronic liver disease

BackgroundPerturbations in iron homeostasis have been reported to be associated with irreversible liver injury in chronic liver disease (CLD). However, it is not clear whether liver dysfunction per se underlies such dysregulation or whether other factors also contribute to it. This study attempted to examine the issues involved.MethodsPatients diagnosed to have chronic liver disease (n = 63), who underwent a medically-indicated upper gastrointestinal endoscopy, were the subjects of this study. Patients with dyspepsia, who underwent such a procedure, and were found to have no endoscopic abnormalities, were used as control subjects (n = 49). Duodenal mucosal samples were obtained to study mRNA and protein levels of duodenal proteins involved in iron absorption. A blood sample was also obtained for estimation of hematological, iron-related, inflammatory and liver function-related parameters.ResultsPatients with CLD had impaired liver function, anemia of inflammation and lower serum levels of hepcidin than control subjects. Gene (mRNA) expression levels of duodenal ferroportin and duodenal cytochrome b (proteins involved in iron absorption) were decreased, while that of divalent metal transporter–1 (DMT-1) was unchanged. Protein expression of DMT-1 was, however, decreased while that of ferroportin was unchanged. In the CLD group, serum hepcidin was predicted independently by serum ferritin and hemoglobin, but not by C-reactive protein (a marker of inflammation). CLD patients with serum ferritin greater than 300 μg/dL had significantly greater liver dysfunction (as indicated by significantly higher serum concentrations of bilirubin, AST and ALT, and MELD scores), higher serum concentrations of CRP and hepcidin, and higher ferroportin protein expression, than those with serum ferritin ≤ 300 μg/dL.ConclusionsIn patients with CLD, anemia of inflammation and low serum hepcidin levels were found to paradoxically co-exist. Expression of duodenal proteins involved in iron absorption were either decreased or unaltered in these patients. The hepcidin response to higher body iron levels and/or inflammation appeared to be functional in these patients, despite the presence of liver disease.     

Mucosal iron binding proteins and the inhibition of iron absorption by endotoxin

The uptake of iron from a tied off jejunal segment into the body after the injection of a 59Fe labeled test dose was decreased after the administration of endotoxin by about 80% in both normal and iron deficient animals.--In the iron deficient group the distribution of 59Fe in the cytosol fraction of jejunal mucosa between transferrin and ferritin was determined chromatographically; the amount of 59Fe in the ferritin fraction increased remarkably after the endotoxin treatment and the ratio of both was changed in favor of ferritin.--It is hypothesized that the association of the diversion of iron to the mucosal ferritin with the decrease of the transport of iron into the blood caused by endotoxin might be the consequence of abnormal oxidations in the mucosa measured by others in liver tissue.