Effect of transfused reticulocytes on iron exchange

A animal model was developed whereby reticulocyte-rich blood was introduced into normal rats by exchange transfusion. Measurements of plasma iron turnover was made at similar plasma iron concentrations before and after exchange transfusions. High reticulocyte blood obtained from animals rendered iron deficient by diet or by treatment with phenylhydrazine resulted in a mean increase of 86% in internal iron exchange, while the plasma iron turnover was unaffected by exchange with normal red cells. Since iron input from reticuloendothelial cells could have increased due to breakdown of transfused cells, iron absorption was also measured. Within 1 hr and for a least 6 hr after exchange with high reticulocyte blood, mean absorption in six groups of animals was increased over control animals by 50%-130%. The increased plasma iron turnover and absorption was not mediated by a decrease in plasma iron or an increase in unsaturated iron-binding capacity. Indeed, a higher plasma iron and transferrin saturation augmented the movement of iron into the plasma from iron- donating tissues. It is proposed that the donation of iron by transferrin in some way immediately facilitates the procurement of more iron by transferrin.     

Transferrin saturation, plasma iron turnover, and transferrin uptake in normal humans

The relationship between plasma iron, transferrin saturation, and plasma iron turnover was studied in 53 normal subjects whose transferrin saturation varied between 17% and 57%, in 25 normal subjects whose transferrin saturation was increased by iron infusion to between 67% and 100%, and in five subjects with early untreated idiopathic hemochromatosis whose transferrin saturation was continually elevated to between 61% and 86%. The plasma iron turnover of all of these subjects ranged from 0.45 to 1.22 mg/dL whole blood/d. The mean values for the above-mentioned three groups were 0.71 +/- 0.17, 1.01 +/- 0.11, and 1.01 +/- 0.13 mg/dL whole blood/d, respectively. Most of this variation, estimated at 72% by regression analysis, was due to a direct relationship between transferrin saturation and plasma iron turnover. This effect was attributed to a competitive advantage of diferric over monoferric transferrin in delivering iron to tissues. This was confirmed by the demonstration of a more rapid clearance of diferric as compared to monoferric transferrin in an additional group of eight normal subjects. Calculations were made of the amount of transferrin reacting with membrane receptors per unit time. Allowance was made for the noncellular (extravascular) exchange and for the 4.2:1 preference of diferric over monoferric transferrin demonstrated in vitro. The amount of iron-bearing transferrin leaving the plasma to bind to tissue receptors for 53 subjects with a transferrin saturation between 17% and 57% was 71 +/- 13; for 25 subjects with a saturation from 67% to 100%, 72 +/- 12; and for five subjects with early idiopathic hemochromatosis, 82 +/- 11 mumol/L whole blood/d. There were no significant differences among these groups. These studies indicate that while the number of iron atoms delivered to the tissues increases with increasing plasma iron and transferrin saturation, the number of iron-bearing transferrin molecules that leave the plasma per unit time to bind to tissue receptors is relatively constant and within the limits studied, independent of transferrin saturation.     

The behavior of transferrin iron in the rat

The behavior of rat transferrin has been investigated employing acrylamide gel electrophoresis and isoelectric focusing. In vitro trace labeling with iron chelates at 30 min was 93%-98% effective, whereas binding by simple ferric salts was reduced to 71%-76%. Complete and specific binding of 59FeSO4 by the iron binding sites of transferrin was demonstrated after in vitro or in vivo addition of ferrous ammonium sulfate in pH 2 saline up to the point of iron saturation. In vitro the radioriron transferrin complex in plasma was stable and its iron had a negligible exchange with other transferrin binding sites over several hours. The distribution of radioiron added in vitro or through absorption was shown to be random between the binding sites of slow and fast transferrin molecule. Iron distribution among body tissues was similar for mono- and diferric transferrin iron and was not affected by the site distribution of iron on the transferrin molecule. The only important aspect of transferrin iron binding was the more rapid tissue uptake of iron in the diferric form was compared to monoferric transferrin. Additional in vivo effects on internal iron exchange were produced by changes in the iron balance of the animal. In the iron loaded animal, monoferric transferrin injected into the plasma was rapidly loaded by iron from tissue and thereby converted to diferric transferrin. Injection of diferric transferrin in the iron deficient animal was associated with a rapid disappearance from circulation of the original complex and a subsequent appearance of monoferric transferrin as a result of iron returning from tissues. These observations support the concept that plasma iron behaves as a single pool except that diferric iron exchange occurs at a more rapid rate than dose monoferric iron exchange.     

Plasma radioiron kinetics in man: explanation for the effect of plasma iron concentration.

The plasma iron turnover was measured in 19 normal subjects. A correlation was found between plasma iron concentration and plasma iron turnover. In addition to the turnover of 55Fe at normal plasma iron concentration (predominantly monoferric transferrin), a second turnover in which the labeled plasma was saturated with iron (to produce predominantly diferric transferrin) was studied with 50Fe. It was demonstrated that diferric transferrin had a greater rate of iron turnover but that the distribution between erythroid and non-erythroid tissues was unchanged. It was concluded that plasma iron turnover is dependent on the monoferric/diferric transferrin ratio in the plasma but that the internal distribution of iron is unaffected.     

Tissue distribution and clearance kinetics of non-transferrin-bound iron in the hypotransferrinemic mouse: a rodent model for hemochromatosis.

Genetically hypotransferrinemic mice accumulate iron in the liver and pancreas. A similar pattern of tissue iron accumulation occurs in humans with hereditary hemochromatosis. In both disorders, there is a decreased plasma concentration of apotransferrin. To test the hypothesis that nontransferrin-bound iron exists and is cleared by the parenchymal tissues, the tissue distribution of 59Fe was studied in animals lacking apotransferrin. Two groups of animals were used: normal rats and mice whose transferrin had been saturated by an intravenous injection of nonradiolabeled iron, and mice with congenital hypotransferrinemia. In control animals, injected 59Fe was found primarily in the bone marrow and spleen. In the transferrin iron-saturated animals, injected 59Fe accumulated in the liver and pancreas. Gastrointestinally absorbed iron in hypotransferrinemic or transferrin iron-saturated mice was deposited in the liver. This indicates that newly absorbed iron is released from mucosal cells not bound to transferrin. Clearance studies demonstrated that transferrin-bound 59Fe was removed from the circulation of rats with a half-time of 50 min. In transferrin iron-saturated animals, injected 59Fe was removed with a half-time of less than 30 s. Analysis of the distribution of 59Fe in serum samples by polyacrylamide gel electrophoresis demonstrated the presence of 59Fe not bound to transferrin. These results demonstrate the existence of and an uptake system for non-transferrin-bound iron. These observations support the hypothesis that parenchymal iron overload is a consequence of reduced concentrations of apotransferrin.     

Quantitation of ferritin iron in plasma, an explanation for non- transferrin iron

In 33 patients with thalassemia and idiopathic hemochromatosis, plasma ferritin protein levels ranged from 36 to 5,850 micrograms/L. The iron content of this ferritin as determined by immunoprecipitation ranged from undetectable amounts to 507 micrograms/L. The mean iron content of ferritin protein in those and other subjects with plasma ferritin concentrations of over 1,000 was 6.8% +/- 2.7%. Plasma transferrin was usually saturated with iron in patients with measurable ferritin iron, but exceptions occurred. In studies using electrophoretic separation, it was shown that some ferritin iron moved to transferrin during in vitro incubation, whereas exchange in the opposite direction was extremely limited. Because some plasma ferritin iron was measured by the standard colorimetric plasma iron determination, these observations (a) indicate that plasma ferritin contains a significant amount of iron (b) indicate that a significant proportion of nontransferrin iron in individuals with nontransferrin iron as detected by standard plasma iron and total iron-binding capacity measurements is due to the presence of ferritin, and (c) suggest that large amounts of ferritin iron may affect the saturation of plasma transferrin.     

The significance of transferrin for intestinal iron absorption

A mechanism is proposed by which apotransferrin is secreted from mucosal cells, loaded with iron in the intestinal lumen, and then the intact complex is taken into the cell. Within the cell, iron is released and transferred to the blood stream, whereas iron-free transferrin returns to the brush border to be recycled. We have investigated this hypothesis by measuring intestinal absorption of radioiron and 125I-labeled plasma transferrin using tied-off gut segments in normal and iron-deficient rats. There was no absorption of diferric transferrin from the ileum, but high absorption from the duodenum and jejunum segments. Jejunal absorption occurred as a function of the dose offered and showed saturation kinetics. In normal animals, 4 micrograms of the 50 micrograms of transferrin iron was absorbed over 1 hr. In iron-deficient animals, mean values as high as 13 micrograms were observed. Radioiron content of the jejunal mucosa bore a linear relationship to the dose administered and was inversely proportional to the amount of iron entering the plasma. Recycling of transferrin was indicated by the presence of labeled apotransferrin in the lumen, first observed between 15 and 60 min after the injection of diferric transferrin. A high resistance of diferric and apotransferrin to proteolytic degradation within the gut lumen was demonstrated. Comparative studies with lactoferrin and ferritin disclosed poor availability of their iron for absorption. The small amount that was absorbed did not relate to the iron status of the recipient animal. These studies support the role of mucosal transferrin as a shuttle protein for iron absorption.     

The Effect of Sex Difference on Iron Exchange in the Rat

S ummary . When compared with normal males, female Wistar rats have significantly higher serum iron levels, a higher rate of plasma iron turnover and a higher proportion of their plasma iron is diverted into non-erythroid tissues. These difference are abolished by castration and reestablished by the supplementation of testosterone and oestrogen to castrated animals. The effect of sex hormones on iron handling is independent of their action on erythropoiesis, since in hypertransfused animals with complete inhibition of erythropoiesis the sex difference in serum iron levels and in hepatic iron uptake is maintained, The absolute rate of iron absorption is similar in both groups, but when the daily absorption of food iron is related to growth rate, it is considerably higher in females than in males (P< 0.001). The cumulative effect of a relatively higher iron absorption in females is the establishment of large iron stores which by virtue of their higher rate of exchange with transferrin contribute to their higher plasma iron turnover.     

The relationship between plasma iron and plasma iron turnover in the rat

Plasma iron turnover has been evaluated in the growing rat. Consistent data were obtained with the intravenous injection of radioiron in the form of ferrous sulfate or ferric citrate. Plasma iron turnover changed as a function of plasma iron concentration. Only part of this effect in the rat was due to the different rates of clearance of mono-and differic transferrin, the latter having a higher iron delivery rate in vivo. An additional effect was shown to relate to the rate of red cell production. With decreased production, the effect of plasma iron on plasma iron turnover was reduced, whereas with increased erythropoiesis there was an additional increment in plasma iron turnover for any increase in plasma iron. Since this effect was observed when increased iron demands were due to an increase in erythroid precursors in the marrow but not in the circulating blood, it is attributed to limitations in iron flow to the marrow. This suggests that erythroid marrow activity and the adequacy of iron supply when studied by ferrokinetic techniques can best be defined by the response curve relating plasma iron concentration to plasma iron turnover.     

Iron Mobilization in the Pregnant Rat

S ummary . Iron exchange in the pregnant rat was quantitated by repeated determinations of plasma iron turnover (PIT), transferrin iron distribution and measurements of storage iron and food iron utilization employing selective radio-iron probes. Despite a sixfold increase in PIT, intestinal absorption of iron accounted for 40% of the PIT throughout pregnancy, with variations not exceeding ±5%. Increased fetal requirements were efficiently compensated for by the mobilization of iron from maternal tissue stores and by increased absorption, and there was no subsequent reduction in iron supply to maternal tissues. Enhancement of iron absorption occurred in the absence of a reduction in serum iron levels or the size of iron stores. In view of the close correlation between PIT and rates of absorption it is postulated that iron absorption in the pregnant rat is regulated by PIT which in turn is determined by the rate of plasma iron clearance by the placenta and the maternal erythroid marrow.     

Role of transferrin in determining internal iron distribution

The behavior in vivo of transferrin in loading and unloading iron from its two sites was examined in rats. Radioiron entering the plasma from the gastrointestinal tract in iron-deficient, normal and iron-loaded rats did not differ in its subsequent tissue distribution between erythroid marrow and liver of normal recipients from a second isotope added to the same plasma in vitro. Loading studies in vitro were then carried out employing a reticulocyte incubation model designed to place one isotope predominantly on one site of transferrin, more available to the erythron, and the second isotope on the other site, more available to the liver. In 15 groups of animals in which 3 different iron salts were employed to load transferrin with iron, the mean isotope ratio in the erythron was 1.03 (+/-0.06 SD) and the mean liver ratio was 0.75 (+/-0.21 SD). It was found that the incubation of plasma with reticulocytes resulted in contamination of the plasma by radioactive hemoglobin. After allowance was made for hepatic uptake of radiohemoglobin in the 13 groups in which proper correction could be made, the isotope ratio in the liver became 0.97 (+/-0.17 SD). It is concluded that iron atoms from the two sites of transferrin have similar tissue distributions in vivo in the experimental situations examined.     

Transferrin: physiologic behavior and clinical implications

The transferrin iron transport system, along with its procurement sites and delivery receptors, provides a highly effective means of satisfying internal iron requirements. Iron uptake by individual tissues is determined by their receptor number, by the relative amounts of monoferric and diferric transferrin in circulation, and by the amount of available iron in donor tissues. Although the modus operandi of this system under basal conditions has been characterized, its exquisite regulation remains an enigma. In some manner, the procurement of iron is determined by iron requirements. What seems to be an inappropriate behavior of the absorptive mechanism in thalassemia and certain other erythroid overload states may actually be life-saving in the absence of transfusion, since it results in higher levels of plasma iron and thereby higher levels of erythropoiesis. The definition of the regulatory mechanism in such conditions may well lead to an understanding of the molecular defect in idiopathic hemochromatosis.     

Anemia of the Belgrade rat: evidence for defective membrane transport of iron

The mechanisms underlying the impaired utilization of transferrin-bound iron by erythroid cells in the anemia of the Belgrade laboratory rat were investigated using reticulocytes from homozygous anemic animals and transferrin labeled with 59Fe and 125I. The results were compared with those obtained using reticulocytes from phenylhydrazine-treated rats and iron-deficient rats. Each step in the iron uptake mechanism was investigated, ie, transferrin-receptor interaction, transferrin endocytosis, iron release from transferrin, and transferrin exocytosis. Although there were quantitative differences, no fundamental difference was found in any of the abovementioned aspects of cellular function when the reticulocytes from Belgrade rats were compared with those from iron-deficient animals. The basic defect in the Belgrade reticulocytes must therefore reside in subsequent steps in iron uptake, after it is released from transferrin within endocytotic vesicles, ie, in the mechanism by which it is transferred across the lining membrane of the vesicles into the cell cytosol. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of reticulocyte ghosts extracts demonstrated a prominent protein band of mol wt 69,000 that was absent or present only in low concentration extracts from the other two types of reticulocytes. This may be a result of the genetic defect.     

Iron uptake from plasma transferrin by the duodenum is impaired in the Hfe knockout mouse

Hereditary hemochromatosis (HH) is a disorder of iron metabolism in which enhanced iron absorption of dietary iron causes increased iron accumulation in the liver, heart, and pancreas. Most individuals with HH are homozygous for a C282Y mutation in the HFE gene. The function of HFE protein is unknown, but it is hypothesized that it acts in association with beta(2)-microglobulin and transferrin receptor 1 to regulate iron uptake from plasma transferrin by the duodenum, the proposed mechanism by which body iron levels are sensed. The aim of this study was to test this hypothesis by comparing clearance of transferrin-bound iron in Hfe knockout (KO) mice with that observed in C57BL/6 control mice. The mice were fed either an iron-deficient, control, or iron-loaded diet for 6 weeks to alter body iron status. The mice then were injected i.v. with (59)Fe-transferrin, and blood samples were taken over 2 h to determine the plasma (59)Fe turnover. After 2 h, the mice were killed and the amount of radioactivity in the duodenum, liver, and kidney was measured. In both Hfe KO and C57BL/6 mice, plasma iron turnover and iron uptake from plasma transferrin by the duodenum, liver, and kidney correlated positively with plasma iron concentration. However, duodenal iron uptake from plasma transferrin was decreased in the Hfe KO mice compared with the control mice. Despite this difference in duodenal uptake, the Hfe KO mice showed no decrease in iron uptake by the liver and kidney or alteration in the plasma iron turnover when compared with C57BL/6 mice. These data support the hypothesis that HFE regulates duodenal uptake of transferrin-bound iron from plasma, and that this mechanism of sensing body iron status, as reflected in plasma iron levels, is impaired in HH.     

Storage Iron Kinetics

S ummary . Iron metabolism in the rat has been examined by chemical and isotopic measurements. Iron stores were larger in the female than the male, but in neither sex did stores play a dominant role in meeting increased iron needs. A nearly two-fold higher plasma iron turnover in growing rats as compared with adult animals was accounted for largely by growth requirements. In both sexes and in both young and adult rats, the erythroid marrow assimilated more than two-thirds of the plasma iron turnover. The source and distribution of transferrin iron was assessed in relation to the erythroid marrow, gastrointestinal tract, hepatic parenchyma and remaining body tissues. Absorption from the gastrointestinal tract provided about one-third of the iron entering the plasma in adult rats and more than half in growing or phlebotomized rats. Iron absorption was closely related to the erythroid marrow requirements, but did not correlate with either the plasma iron level or tissue iron stores. The rat depends on dietary iron rather than iron stores to meet increased requirements and shows a much greater capacity to absorb iron than does man.     

Targeted gene disruption reveals an essential role for ceruloplasmin in cellular iron efflux

Aceruloplasminemia is an autosomal recessive disorder of iron metabolism. Affected individuals evidence iron accumulation in tissue parenchyma in association with absent serum ceruloplasmin. Genetic studies of such patients reveal inherited mutations in the ceruloplasmin gene. To elucidate the role of ceruloplasmin in iron homeostasis, we created an animal model of aceruloplasminemia by disrupting the murine ceruloplasmin (Cp) gene. Although normal at birth, Cp(-/-) mice demonstrate progressive accumulation of iron such that by one year of age all animals have a prominent elevation in serum ferritin and a 3- to 6-fold increase in the iron content of the liver and spleen. Histological analysis of affected tissues in these mice shows abundant iron stores within reticuloendothelial cells and hepatocytes. Ferrokinetic studies in Cp(+/+) and Cp(-/-) mice reveal equivalent rates of iron absorption and plasma iron turnover, suggesting that iron accumulation results from altered compartmentalization within the iron cycle. Consistent with this concept, Cp(-/-) mice showed no abnormalities in cellular iron uptake but a striking impairment in the movement of iron out of reticuloendothelial cells and hepatocytes. Our findings reveal an essential physiologic role for ceruloplasmin in determining the rate of iron efflux from cells with mobilizable iron stores.     

Occupancy of the iron binding sites of human transferrin.

The in vivo distribution of iron between the binding sites of transferrin was examined. Plasma was obtained from normal subjects under basal conditions and after in vitro and in vivo iron loading. Independent methods, including measurement of the transferrin profile after isoelectric focusing and cross immunoelectrophoresis, and determination of the iron content in the separated fractions were in agreement that there was a random distribution of iron on binding sites. This held true with in vitro loading, when iron was increased by intestinal absorption and with loading from the reticuloendothelial system. The data indicate that the distribution of apo-, monoferric, and diferric transferrins is predictable on the basis of the plasma transferrin saturation and negate the concept that iron loading of transferrin in vitro is a selective process with possible functional consequences in tissue iron delivery.     

In vitro and in vivo studies of iron delivery by human monoferric transferrins

S ummary . According to the Fletcher-Huehns hypothesis there exists a functional difference between the two iron-binding sites of transferrin. In this study we present the results of an evaluation of this hypothesis in vitro and in vivo with human pure monoferric transferrins obtained by preparative isoelectric focusing in granulated gels. The uptake of iron from monoferric transferrins TfFe_c and Fe_NTf by erythroid bone marrow cells, hepatocytes and stimulated T-lymphocytes in vitro was equal, even when both monoferric transferrins were present together in the incubation medium. Ferrokinetic studies in vivo , performed with both pure monoferric transferrins, showed that transferrin TfFe_c, as well as transferrin Fe_NTf, mainly deliver their iron to the erythron. As red cell ⁵⁹Fe utilization, red cell iron turnover and other ferrokinetic parameters, obtained from this study, were identical too it is evident that both iron-binding sites of transferrin are functionally homogeneous in vivo , with respect to iron delivery.     

Cellular iron processing

Iron is transported in the blood plasma, mainly bound to transferrin, but in abnormal conditions other iron containing compounds may become important. These include ferritin, haemopexin-haem, haptoglobin-haemoglobin and non-specific non-transferrin-bound iron, all of which are taken up from the circulation by the liver. Transferrin-bound iron can be used by all types of cells in amounts that depend on their complement of transferrin receptors. Immature erythroid cells are the most active in this function. Investigations using reticulocytes as an example of erythroid cells have demonstrated the presence of two mechanisms for the uptake of ferrous iron. One, a high affinity process disappears as reticulocytes mature. It probably represents the mechanism by which iron derived from transferrin is transported into the cytosol after receptor-mediated endocytosis of the iron-transferrin complex. The other mechanism has a lower affinity for iron, is retained when reticulocytes mature and is probably associated with Na⁺ transport across the cell membrane. The physiological characteristics of the two iron transport processes and the evidence for the above conclusions are summarized in the present paper.