Iron-deficient Animals Research Articles

Iron binding proteins of iron-absorbing rat intestinal mucosa.

The distribution and quantitation of the iron-binding proteins of rat small intestinal mucosa was studied, in iron-deficient and replete animals, to explore their role in the absorption of iron. Adsorption (mucosal uptake) of iron in in situ ligated loops of small intestinal mucosa was found to be uniform throughout the length of the small intestine whereas absorption (carcass uptake) showed a steep decreasing gradient from the duodenum to the ileum. The disrupted, in vivo labeled mucosal cells were fractionated by isopycnic centrifugation and transferrin and ferritin were quantitated by radioimmunoassay. Transferrin derived from mucosal cells was shown to have a higher affinity for the antibody than transferrin in serum. Of the transferrin present in the mucosal extract, only a portion could be accounted for by contamination from the serum; the proteolysis resistant and intrinsic transferrin may be mucosal cell specific. Transferrin was found in similar amounts in all regions of the small intestine, was not affected by iron loading but doubled in response to iron deficiency. Mucosal ferritin was found in greater amounts in the iron-absorbing areas of the intestine, increased in the duodenum of iron-loaded animals, and decreased in iron-deficient animals. The incorporation of newly absorbed radioiron into ferritin was only found in iron absorbing regions and was completely inhibited by colchicine and cytochalasin-B, suggesting that ferritin was loaded with iron at the point of iron absorption and that the process is associated with vesicle movement and not simple diffusion. Transferrin and ferritin-specific immunoabsorption and also gel filtration established that no other soluble iron binding proteins were involved in absorption.

The effect of iron deficiency on experimental oral carcinogenesis in the rat.

The effect of iron deficiency on oral carcinogenesis was investigated in 30 young adult male Charles River white rats. In 15 animals, prior to the start of carcinogen treatment, iron deficiency anaemia was produced and subsequently maintained by a combination of low iron diet (12 mg Fe2+ kg-1 diet) and repeated venesection. Fifteen control animals were fed the same diet supplemented with iron to approximately 140 mg FE2+ kg-1 diet. All animals were treated with the carcinogen 0.5% 4-Nitroquinoline-N-oxide in propylene glycol which was painted on the palate 3 times weekly. Animals were killed when tumours were grossly evident. The mean haemoglobin levels at the start of carcinogen applications were 10.1 g dl-1 in the anaemic group and 14.1 g dl-1 in the control group, and at the time of killing were 8.2 g dl-1 in anaemic animals and 13.8 g dl-1 in controls. The incidence of animals developing squamous cell carcinomas was similar in both groups, but tumour development was significantly earlier in iron-deficient animals (mean 183 days) compared to controls (mean 229 days). Iron-deficient animals showed a significantly greater incidence of tongue tumours and control animals showed a significantly greater incidence of palatal tumours.

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 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.

Distribution and metabolism of iron in muscles of iron-deficient rats

Iron-deficiency anemia leads directly to both reduced hemoglobin levels and work performance in humans and experimental animals. In an attempt to observe a direct link between work performance and insufficient iron at the cellular level, we produced severe iron deficiency in female weanling Sprague-Dawley rats following five weeks on a low-iron diet. Deficient rats were compared with normal animals to observe major changes in hematological parameters, body weight, and growth of certain organs and tissues. The overall growth of iron-deficient animals was approximately 50% of normal. The ratio of organ weight: body weight increased in heart, liver, spleen, kidney, brain, and soleus muscle in response to iron deficiency. Further, mitochondria from heart and red muscle retained their iron more effectively under the stress of iron deficiency than mitochondria from liver and spleen.Metabolism of iron in normal and depleted tissue was measured using tracer amounts of(59)Fe administered orally. As expected, there was greater uptake of tracer iron by iron-deficient animals. The major organ of iron accumulation was the spleen, but significant amounts of isotope were also localized in heart and brain. In all muscle tissue examined the(59)Fe preferentially entered the mitochondria. Enhanced mitochondrial uptake of iron prior to any detectable change in the hemoglobin level in experimental animals may be indicative of nonhemoglobin related biochemical changes and/or decrements in work capacity.

Changes in corticosterone levels in iron deficient rats

The hypothesis that behavioural changes in iron deficiency are a product of stress was tested by monitoring corticosterone levels. Adult hooded rats were placed on an iron deficient diet for 2 (Stage 1), 8 (Stage 2) or 12 (Stage 3) weeks. When trained and tested on a single-trial taste aversion task, iron deficient animals showed the same performance as seen in previous experiments. Poor performance in early iron deficiency were followed by a temporary return to normal levels as the deficiency progressed, and falling below normal with continued iron depletion. Iron deficient animals failed to demonstrate the predicted stress-related changes in cortocosterone levels. Instead of an increasing in corticosterone levels after conditioning, levels fell to well below normal at all stages of deficiency following conditioning. Iron deficiency probably limits the action of certain enzymes responsible for corticosterone production. Due to the similarity of changes in catecholamines in stress and iron deficiency, it is possible that these changes will explain the pattern of behavioural response in iron depletion.

Influence of dietary iron deficiency on hemoglobin, myoglobin, their respective reductases, and skeletal muscle mitochondrial respiration

Male weanling rats were fed a control diet (46 ppm iron) or an iron-deficient diet (11 ppm iron) for 7 wk to determine the influence of iron deficiency on heme proteins and skeletal muscle mitochondrial respiration. At the end of 7 wk, the hemoglobin in the blood of the iron deficient rats was 35% less and skeletal muscle myoglobin was 20 to 37% less than in the control animals. The concentration of myoglobin in the heart was not appreciably diminished by iron deficiency. Cytochrome c concentration was 20% less in the heart and 35% less in the mixed-fiber gastrocnemius in the iron-deficient animals. Iron deficiency did not influence the activity of metmyoglobin reductase in either heart or skeletal muscle. There was about 30% more methemoglobin reductase activity in the red blood cells of the iron-deficient animals, which resulted in methemoglobin levels that were so low as to be virtually unmeasurable. In the iron-deficient rats, skeletal muscle mitochondrial respiration with either pyruvate-malate or palmitylcarnitine as substrate was 17 to 20% less than in the control animals. This study demonstrates that dietary iron deficiency of sufficient severity to reduce blood Hb and skeletal muscle myoglobin or cytochrome c also results in an impaired skeletal muscle oxidative capacity. The study also illustrates the preferential utilization of iron, not only between tissues, but within tissues, and tissue specific adaptive responses to iron deficiency.

Determination of transferrin-like immunoreactivity in the mucosal homogenate of the duodenum, jejunum, and ileum of normal and iron deficient rats.

1. In intestinal mucosal tissue a transferrin-like immunoreactivity (TLIR) was determined after loading with 59Fe-(FeCl3) in vivo. The scraped-off mucosal tissue was homogenized and centrifuged at 100 000 X g. The supernatant was fractionated chromatographically. The TLIR was determined by the Mancini-test. 2. Extrapolated to infinite dilutions of the homogenate the following contents of the TLIR were calculated per cm of the intestinal segment: 28, 20, and 13, microgram/cm in the duodenum, jejunum, and ileum of iron-deficient animals and 15, 14, and 12 microgram/cm in the corresponding intestinal segments of normal rats, respectively. 3. The content of TLIR was compared with the iron uptake of the mucosal tissue from the intestinal lumen during absorption. An "iron turnover number" of 1.6 +/- 0.2 iron atoms per min during the absorption in duodenal and jejunal segments and of 0.5 +/- 0.1 in ileal segments of either iron-deficient and normal animals was calculated for the mucosal transferrin represented by the TLIR.

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.

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.

Hypothermia in iron deficiency due to altered triiodothyronine metabolism.

Iron-deficient rats become hypothermic and have an excessive catecholamine response when exposed to an ambient temperature of 4 degrees C. This is not due to changes in body insulation, since thickness is unaltered, since differences persist after removal of hair, and since cutaneous vasoconstriction is intact. On the other hand, oxygen consumption of iron-deficient animals at 4 degrees C is reduced, 39 +/- 3 ml . kg-1 . min-1 compared to 63 +/- 2 in control animals. Thyroxine (T4) values at 4 degrees C were 4.34 +/- 0.20 microgram/dl sera as compared to control values of 3.6 +/- 0.32. Triiodothyronine (T3) values of iron-deficient animals in the cold were 48 +/- 6.8 ng/dl as compared to 72 +/- 5.6 in control animals. Treatment of iron-deficient animals with iron was shown to normalize the plasma T3 response at 4 degrees C within 6 days. Thyroidectomized iron-deficient animals injected with T3 did not show hypothermia at 4 degrees C, whereas thyroidectomized iron-deficient animals injected with T4 showed hypothermia, increased catecholamines, and decreased T3 levels as compared to non-iron-deficient animals similarly treated. It is proposed that iron deficiency impairs conversion of T4 to T3 and that this is primarily responsible for the hypothermia observed.

Iron deficiency during early development in the rat: Behavioral and physiological consequences

An iron deficient diet regimen from birth through 28 to 30 days of age produced animals with decreases levels of brain non-heme iron as well as decreased brain weight and hematocrit. Weight gain was also somewhat slower. In contrast, brain levels of cytochrome c were not reduced. During subsequent testing iron deficient animals were less responsive than controls in a mildly aversive novel situation (the open field), and ambulated less in an exploratory task (the hole-board). Iron deficient males also exhibited longer response latencies when first exposed to the passive avoidance platform, but following shock, iron deficient animals of both sexes exhibited longer reentry latencies. Measurement of plasma levels of corticosterone indicated that although iron deficient animals had elevated basal levels of corticoids, they exhibited a smaller stress increment than controls when exposed to the combined stress of ether and cardiac puncture. Data from the three behavioral tasks taken together with the pituitary-adrenal response to ether and cardiac puncture suggest that iron deficiency may reduce an animal's general responsiveness to environmental stimuli.

Interrelationships Between Iron and Lead Absorption in Iron-Deficient Mice

Mice were made iron-deficient by feeding a semisynthetic low-iron diet, by bleeding, or by a combination of both methods. Dietary-induced iron deficiency, by itself, enhanced iron absorption from an. intragastric dose to a much greater extent than did bleeding mice fed an iron-supplemented diet. Enhanced lead absorption was seen in the former but not in the latter group. Bleeding was effective, however, in raising lead absorption when mice were fed the iron-deficient diet. Additionally, the kinetics of lead absorption were studied by using an openended duodenal perfusion technique. When lead was perfused in solutions containing either 0.15 M sodium chloride or 0.15 M sodium ascorbate, irondeficient animals took up more lead into the mucosa and transferred more to the body than mice fed an iron-supplemented diet. Intestinal uptake of lead from both perfusates in iron-sufficient animals appeared to occur by a process that was primarily dependent on the luminal lead concentration. Lead uptake from the saline perfusate in iron-deficient mice was also directly related to the lead concentration of the perfusate, whereas uptake from the ascorbate perfusate was proportionally greater from lowerthan from higher-lead concentrations, giving rise to saturation-type kinetics. Lead transfer to the body from both types of perfusate also displayed saturation kinetics in both iron-sufficient and iron-deficient animals. When iron and lead were perfused together in 0.15 M sodium ascorbate, iron inhibited lead up take and transfer at Fe/Pb molar ratios as low as 0.5:1, whereas lead inhibited iron uptake and transfer at a Pb/Fe ratio of 5:1. These mutually inhibitory effects were not as easily demonstrated with a saline perfusate. Our results suggest that lead absorption occurs by two simultaneous processes: the first is a diffusion-type mechanism, and the second is a carrier-mediated process, which is enhanced by feeding a low-iron diet. Lead appeared to have less affinity than iron for the latter mechanism.

Catecholamine elevation in iron deficiency.

Iron-deficient rats have increased blood and urinary catecholamines regardless of whether anemia is or is not present. The catecholamine response in both iron-deficient and control animals is largely temperature dependent, showing little difference at the isothermic temperature of 30 degrees C but a two- to threefold increase in iron-deficient animals over controls at lower temperatures. The iron-deficient rat is unable to maintain body temperature at 4 degrees C and this is independent of anemia or of food intake. When animals are run on the treadmill for 4 h, body temperatures increase but the difference observed at 4 degrees C between iron-deficient and control animals persists. The underlying abnormality in temperature regulation and in catecholamine response disappeared after 6 days of iron therapy.

Iron deficiency in the rat: effects on phenylalanine metabolism.

Concentrations of phenylalanine in the plasma were markedly elevated in iron-deficient rats and appeared to vary directly with the degree of iron deficiency. Plasma concentrations of phenylalanine returned to control levels within one week after treatment of the iron-deficient rats with iron dextran. The elevated levels of plasma phenylalanine were probably not produced by a deficiency in liver phenylalanine hydroxylase because levels of activity of the enzyme were found to be normal in the livers of the iron-deficient animals.

Lactic Acidosis as a Result of Iron Deficiency

Iron-deficient rats have an impaired work performance, even when their anemia is corrected by exchange transfusion. Muscle activity is associated with a higher blood lactate concentration than is observed in iron-replete animals. The accumulation of lactate is a result of excessive production as lactate clearance from the blood was shown to be unaffected. By adjusting the work load to a lower level, it was possible to divide iron-deficient animals into two groups, one capable of continued treadmill running and another in which animals stopped before 20 min. In the former, blood lactate concentration reached a plateau at moderate levels, whereas it continued to increase in the latter until the animal stopped running. Levels of alpha-glycerophosphate oxidase in skeletal muscle mitochondria were found to be much lower in the second group (P < 0.001). Lactate infusion into normal animals was shown to interfere with work performance, and maintenance of a normal pH in iron-deficient and iron-replete animals did not prevent the impairment in work associated with high blood lactate concentrations. Additional evidence was obtained that energy substrate (blood glucose and free fatty acids, muscle glycogen) was adequate in irondeficient animals. Oxygen tension in their vena caval blood was higher than in controls. Furthermore, the in situ behavior of electrically stimulated gastroenemius and soleus muscles appeared similar to that of control animals. Because the stimulation of the single muscle in the iron-deficient animal did not result in appreciable elevation of blood lactate and did not show impaired contractility further supported the hypothesis that the elevation of blood lactate caused the decreased work performance. It is concluded that iron deficiency by a depletion in the iron-containing mitochondrial enzyme, alpha-glycerophosphate oxidase, impairs glycolysis, resulting in excess lactate formation, which at high levels leads to cessation of physical activity.

Iron Deficiency Prevents Liver Toxicity of 2,3,7,8-Tetrachlorodibenzo- p -Dioxin

The compound 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) causes hepatocellular damage and porphyria in C57B1/6J mice, among a wide range of toxic effects. We compared the effect of TCDD toxicity in iron-deficient mice with that in mice receiving a normal diet. Porphyria did not develop in the iron-deficient animals, and these animals were also protected from hepatocellular damage and certain other toxic effects of TCDD.

Iron absorption by rats from nonprescription dietary iron supplements.

Absorption of iron from seven nonprescription dietary iron supplements was measured in both iron-replete and iron-depleted rats by use of the extrinsic label technique. Both ferrous and ferric forms of the radioactive extrinsic tracer iron were used; the iron was in the form of 59FeS04, 59FeC13, or ferr~us-~~Fe gluconate. Of six products tested with both ferrous and ferric labels, significant differences in absorption measured by the two different labels were found for three products. This suggests that there is incomplete exchange between the intrinsic and extrinsic iron pools. Iron-deficient animals absorbed significantly more iron than iron replete animals when the ferrous label was used in both experiments. Although iron-replete animals discriminate among iron sources, absorption is always less than in iron-deficient animals. The results with iron deficient animals show the true bioavailability of the iron. The use of the extrinsic label may not always be valid when the metal in question can exist in more than one oxidation state. The absorption of iron from various food products has been estimated by use of an extrinsic radioactive iron tracer (Bjorn-Rasmussen et al., 1972, 1973; Bjorn-Rasmussen, 1973; Monsen, 1974). The extrinsic iron exchanges with the intrinsic non-heme iron of the food and can thus be used to determine absorption. However, much of the iron in some foods, especially breads and cereals, is present as inorganic iron which is added as fortification, rather than being biosynthetically incorporated into the food. The use of supplementary iron salts in tablet form is widespread. We wished to examine the use of the extrinsic label method to measure absorption of such inorganic iron which is not necessarily in the same chemical form as the radioactive tracer. We chose to examine iron absorption from a number of over-the-counter dietary iron supplements that contained iron in various forms and varying amounts of vitamins, other minerals, binders, sweeteners, and alcohol. Absorption was measured in iron replete rats with the use of radioactive tracers in both the ferrous and ferric state and in iron-deficient animals with the use of a ferrous label. MATERIALS AND METHODS Iron supplements were purchased from local supermarkets and drug stores. Solubility of the iron in the nonliquid iron supplements was measured in HC1 and in water. One tablet of a supplement was placed in 25 mL of 1.5 M HCl and shaken at 37 “C for 18 h. Disintegration of the tablets was checked at 5-minute intervals for 1 h, at 2 h and at 18 h. After 18 h, any large particles remaining were pulverized. One hour later the solutions were filtered, and iron was determined by atomic absorption. We

Factors affecting the absorption and excretion of lead in the rat

A reliable method for studying lead absorption and excretion in rats is described. Lead absorption occurs primarily in the duodenum where lead enters the epithelial mucosal cells. There is a relative mucosal block for lead with increasing intraluminal doses. Certain substances which bind lead and increase its solubility enhance its absorption. Iron, zinc, and calcium decrease the absorption of lead without affecting its solubility, probably by competing for shared absorptive receptors in the intestinal mucosa. The total body burden of lead does not affect lead absorption. Thus, lead does not have a feedback mechanism which limits absorption. Lead absorption is increased during rapid periods of growth and in iron-deficient animals. It is diminished with starvation and in iron-overloaded animals. The excretion and kinetics of tracer doses of radiolead were quantified. Erythrocytes seem to serve an important role in transport. Excretion occurs in urine and stool. Bile is an important route of excretion in the gut. Although most of a tracer dose is rapidly excreted, the excretory process is limited permitting lead accumulation primarily in bone.

Lysozyme levels in tissues of iron-deficient rats.

Fats were fed either diets sufficient (300 ppm) or insufficient (5 ppm) in iron for 10 weeks. The iron-deficient animals had lowered hemoglobin and hematocrit levels and higher levels of kidney lysozyme activity than did control animals. There were no significant changes in serum and spleen lysozyme activity levels.