Iron-deficient Rats Research Articles

Absorption of 233U, 237Np, 238Pu, 241Am and 244Cm from the gastrointestinal tracts of rats fed an iron-deficient diet.

Absorption of U, Np, Am and Cm was increased by factors of 3.4, 7.1, 2.7 and 1.7, respectively, when nitrate solutions of these actinides were gavaged to adult rats fed an iron-deficient diet. Retention increased proportionately in liver, kidney and carcass. The concentration of the actinides excreted also increased substantially (over that of controls) in the urine of iron-deficient rats gavaged with 233U and 237Np, but not in those with 241Am or 244Cm. Weanling rats on an iron-deficient diet, gavaged with ferric nitrate immediately before administration of 238Pu nitrate, retained between 4% and 12% of the 238Pu retained by litter mates that were not treated intragastrically with iron.

On the origin of intestinal transferrin.

The incorporation of 35S-L-methionine (35S-Met) into TCA-precipitable protein is used to measure protein synthesis in isolated non-vascular perfused jejunal segments and in isolated liver cells under steady-state conditions in rats. 10(5) X g supernatants of homogenates from jejunal segments and from liver cells as well as the jejunal absorbate were processed immuno-electrophoretically. Incorporation of 35S-Met radioactivity into precipitin lines with sera against transferrin, IgG and plasma proteins were autoradiographed and compared semiquantitatively with each other. Calculated on a wet-weight basis this system is sensitive enough to detect transferrin synthesis down to a level of 1% of that in the liver. Still, no transferrin synthesis was found in the jejunal mucosa, while 35S-Met incorporation into TCA precipitates and into IgG continued in isolated jejunal segments for over 2 h. A good correlation was found (r = 0.88, P less than 0.01) between mucosal and plasma transferrin in normal as well as in iron deficient rats. A complete immunologic cross-reactivity could be demonstrated between different plasma transferrins and the transferrin in three different preparations of the intestinal mucosa. Immunoblots of electropherograms after isoelectric focussing showed no distinct differences between transferrin in the plasma, bile, and in the mucosal epithelium.

Experimental study on effects of iron deficiency anemia on immune function.

Weanling rats and mice were fed on a low-iron diet. By the end of the 4th week, the rats were sacrificed and tested for state 01 immunity, and the mice were infected with S. typhimurium. Rate of peripheral blood lymphocytic transformation, total serum complement activity (CH50) and splenic tissue cAMP levels in the iron-deficient rats were significantly decreased as compared with those of the controls. There was no significant difference in the levels of serum hemolysin (HC50) and serum lysozyme between iron-deficient and control rats. Gross histological changes could be observed in the spleen of the iron-deficient rats, but no change was seen in the thymus. After intraperitoneal injection with S. typhimurium, the mortality of iron-deficient mice was significantly increased as compared with that of the controls.

Induction of an increase in mitochondrial matrix enzymes in muscle of iron-deficient rats.

Young rats maintained on an iron-deficient diet developed severe anemia and had large decreases in the levels of the iron-containing flavoproteins and cytochromes of the mitochondrial respiratory chain in skeletal muscle. In contrast, the levels of a number of mitochondrial matrix marker enzymes, including citrate synthase, isocitrate dehydrogenase, 3-hydroxyacyl-CoA dehydrogenase, 3-ketoacid-CoA transferase, and aspartate aminotransferase, increased in red skeletal muscle but not in white muscle. Phosphocreatine concentration was decreased and inorganic phosphate concentration was increased in soleus muscle frozen in situ. We hypothesize that the increase in mitochondrial matrix enzymes reflects a stimulus to mitochondrial biogenesis in posture-maintaining and weight-bearing red muscle fibers in severely iron-deficient rats. It is our working hypothesis that this stimulus to mitochondrial biogenesis arises from mild activity of the red fibers and is due to the same perturbation in cellular homeostasis that is normally caused by vigorous exercise or hypoxia. In iron deficiency, the stimulus to mitochondrial biogenesis can induce an increase in only those enzymes not prevented from increasing by iron deficiency, resulting in formation of mitochondria of grossly abnormal composition.

Increased glucose dependence in resting, iron-deficient rats.

Rates of blood glucose and lactate turnover were assessed in resting iron-deficient and iron-sufficient (control) rats to test the hypothesis that dependence on glucose metabolism is increased in iron deficiency. Male Sprague-Dawley rats, 21 days old, were fed a diet containing either 6 mg iron/kg feed (iron-deficient group) or 50 mg iron/kg feed (iron-sufficient group) for 3-4 wk. The iron-deficient group became anemic, with hemoglobin levels of 6.4 +/- 0.2 compared with 13.8 +/- 0.3 g/dl for controls. Rats received a 90-min primed continuous infusion of D-[6-3H]glucose and sodium L-[U-14C]lactate via a jugular catheter. Serial samples were taken from a carotid catheter for concentration and specific activity determinations. Iron-deficient rats had significantly (P less than 0.05) higher blood glucose (7.1 +/- 0.3 vs. 6.1 +/- 0.2 mM) and lactate concentrations than controls (1.0 +/- 0.1 vs. 0.8 +/- 0.1 mM). The iron-deficient group had a significantly higher glucose turnover rate (67 +/- 2 vs. 58 +/- 4 mumol . kg-1 . min-1) than the control group. Significantly more metabolite recycling in iron-deficient rats was indicated by greater incorporation of 14C (from infused [14C]-lactate) into blood glucose. Assuming a carbon crossover correction factor of 2, half of blood glucose arose from lactate in deficient animals. By comparison, only 25% of glucose arose from lactate in controls. Lack of a difference in lactate turnover (irreversible disposal) rates between deficient rats and controls (191 +/- 26 vs. 163 +/- 15 mumol . kg-1 . min-1) was attributed to 14C recycling.(ABSTRACT TRUNCATED AT 250 WORDS)

Iron Intake Influences Essential Fatty Acid and Lipid Composition of Rat Plasma and Erythrocytes

In view of the clinical importance of iron deficiency as well as the known role of iron in stearic acid desaturation, the effects of higher or lower iron intake on fatty acid composition of blood and liver in the rat were studied. Male Sprague-Dawley rats were fed purified diets that contained iron at 12, 27 or 237 mg/kg. After 12 wk the lipid and fatty acid composition of plasma, erythrocytes and liver was analyzed. Linoleic acid in plasma phospholipids and triacylglycerols was higher, but arachidonic acid was lower in the group fed 12 mg/kg iron than in the groups fed 27 or 237 mg/kg iron. Liver fatty acid and lipid composition was not different between groups. In the group fed 237 mg/kg iron, plasma cholesterol and triacylglycerols were 150%, erythrocyte cholesterol was 137% and erythrocyte phospholipids were 148% of levels in the rats fed 27 mg/kg iron. The fatty acid data suggest a mild impairment in essential fatty acid metabolism in moderately iron-deficient rats. The lipid data suggest a significant alteration in the total lipid content of plasma and erythrocytes of iron-supplemented rats.

Iron deficiency and interleukin 1 production by rat leukocytes

The production of the immunotransmitter, interleukin 1 (IL-1), by peritoneal exudate cells (PEC) was examined in iron-deficient and control rats. Three groups of weanling male Sprague-Dawley rats were fed a purified diet containing 6, 12, or 35 ppm iron for 6 wk to produce severe iron deficiency, moderate iron deficiency, or adequate iron status. Crude IL-1 samples were prepared from acute PEC and assayed for activity. IL-1 preparations from severely and moderately iron-deficient rats enhanced mouse thymocyte proliferation in vitro less than half as much as IL-1 preparations from control rats. In a rabbit bioassay, injection of IL-1 prepared with PEC from either group of iron-deficient rats had little effect on body temperature or plasma minerals, while IL-1 from iron-adequate source PEC produced a febrile response and markedly lowered plasma iron and zinc in recipient rabbits. Severe or moderate iron deficiency, then, clearly impairs IL-1 production by rat PEC.

SIgA- and IgM-containing cells in the intestinal mucosa of iron-deficient rats

The effect of iron deficiency on the jejunal mucosa was studied in postweaning rats that had received a 3-wk regimen of either iron-deficient or iron-sufficient diet (iron content 6 and 50 mg/kg diet) and in rats given the iron-sufficient diet for 1 wk after the initial 3-wk iron-deficient diet. Morphometric analysis showed little difference in villous height but a significant decrease in mitotic index of the crypt epithelial cells in the iron-deficient group. Direct immunoperoxidase studies showed that iron-deficient rats had substantially fewer sIgA- and IgM-containing cells than iron-sufficient rats. This abnormality was reversed after a 1-wk iron-sufficient diet. We conclude that iron deficiency may impair local immunity in the intestinal mucosa, sensitizing the surface epithelial cells to damage by noxious agents. Similar changes might lead to the syndrome of iron-deficiency anemia and hypoproteinemia in children.

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.

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.

Effects of iron deficiency and training on mitochondrial enzymes in skeletal muscle.

We measured mitochondrial enzyme activities in skeletal muscle under conditions of iron deficiency and endurance training to assess the effects of these interventions on the contents and proportions of non-iron-containing and iron-dependent enzymes and proteins. Male Sprague-Dawley rats, 21 days of age, received a diet containing either 6 (iron deficient) or 50 mg iron/kg diet (iron sufficient). At 35 days of age animals were subdivided into sedentary and endurance training groups (running at 0.7 mph, 0% grade, 45 min/day, 6 days/wk). By 70 days of age, iron deficiency had decreased gastrocnemius muscle cytochrome c by 62% in sedentary animals. In contrast, the activities of tricarboxylic acid cycle enzymes were increased, remained unchanged or were slightly decreased, indicating that iron deficiency markedly altered mitochondrial composition. Endurance training increased cytochrome c (35%), tricarboxylic acid cycle enzymes (approximately 15%), and manganese superoxide dismutase (33%) in iron-deficient rats, whereas the same exercise regimen had no effect on the skeletal muscle of iron-sufficient animals. The interactive effect of dietary iron deficiency and mild exercise on mitochondrial enzymes suggests that adaptation to a training stimulus is, to some extent, geared to the relationship between the energy demand of exercise and the capacity for O2 transport and utilization.

The cadmium effect on iron absorption

Test solutions of cadmium and labeled iron salts, soluble complexes of diferric transferrin, or hemoglobin iron were introduced orally or were injected into tied-off jejunal segments in rat. Cadmium reduced the absorption of iron salts to about half in both normal and iron-deficient rats. Hemoglobin iron absorption was enhanced, indicating that the processing of this form or iron and its release from mucosa to blood was intact. A greater reduction in iron absorption occurred in iron-deficient rats when transferrin iron was injected into gut loops. Mucosal radioiron content in animals given cadmium with either iron salts or transferrin iron was increased. The primary effect of cadmium was on intracellular processing of iron salts and transferrin iron. The major portion of cadmium taken up by the mucosa of normal animals was bound to ferritin, and the effect of cadmium within the mucosal cell may be reduced thus.

Feed efficiency and norepinephrine turnover in iron deficiency.

Norepinephrine turnover and energetic efficiency studies were conducted in three groups of male Sprague-Dawley rats placed on low iron diets for 5 weeks on weaning. Iron-deficient rats had significant anemia (hematocrit less than 20%) and growth retardation relative to pair-fed and ad libitum fed controls who received the same diet plus weekly iron dextran injections. Energetic efficiency over a 7-day period was nearly 30% less in anemic animals. This was associated with significantly higher rates of norepinephrine turnover in brown adipose tissue (110%) and heart (330%) with significant hypertrophy in both tissues. There was no difference in body composition in ad libitum groups. Plasma triiodothyronine and thyroxine were reduced by 37% in iron deficients compared to controls. Thus 39% increase in caloric requirements in iron deficiency is associated with increased sympathetic and perhaps thermogenic activity in brown adipocytes.

Impaired Natural Killer Cell Activity in Iron-Deficient Rat Pups

Natural killer (NK) cell activity was studied in iron-deficient rat pups. Pregnant dams were fed diets containing 6, 10 or 250 ppm Fe ad libitum from d 1 of gestation through d 21 of lactation. Two days post parturition litters were adjusted to seven pups each, and on d 17 the pups were injected intraperitoneally with 5 × 105 plaque-forming units of vaccinia virus. Following a 4-d incubation period, spleens were removed and the cell suspensions combined with YAC-1 target cells to measure cytolysis in a 4- and 16-h chromium release assay. Hematocrit levels of severe (6 ppm) and moderately (10 ppm) iron-deficient rat pups were significantly lower than that of controls. Similarly, body weight and spleen weights were significantly lower in irondeficient pups than in control pups. Iron deficiency significantly impaired spleen NK cell activity when measured by two different effector:target ratios and assay time periods.

Iron deficiency and hearing loss. Experimental study in growing rats.

Cochlear changes were studied in 141 growing rats raised on a basic iron-deficient diet for 7-100 days; 130 rats served as normal or chronic anemia controls. Electrophysiological findings showed that the incidence of an auditory threshold elevation of more than 15 dB was 31.85% in the iron-deficient rats, but it was unchanged in all the control animals. The main cochlear histopathological changes induced by iron deficiency were strial atrophy and reduction of spiral ganglion cells. It is concluded that the observed anomalies may be attributed solely to iron deficiency of the cochlear tissue.

A scanning electron microscopic study of cochlear changes in iron-deficient rats

The effects of iron deficiency on the cochlea were studied in growing rats fed with a basic iron-deficient diet for 80 days. The electrophysiological changes (auditory thresholds raised more than 15 dB) were observed in 47% of the cochleas of iron-deficient rats. When these organs of Corti were examined by scanning electron microscopy, abnormalities of outer or inner hair cells were found, as follows: (1) fusion and torsion of the stereocilia, (2) coalescence of adjacent stereocilia in the same row, (3) loss of sensory hair stiffness, and (4) loss of stereocilia. Within each lesion, the neighbouring supporting cells and their microvilli showed no damage. The findings indicate that cochlear impairment can be induced by iron deficiency. The peroxidative mechanisms responsible for the lesions of stereocilia in iron deficiency are discussed.

Alteration of intracellular synthesis of surface membrane glycoproteins in small intestine of iron-deficient rats.

We studied the activity and kinetic parameters of microvillous enzymes in intestinal villous cells and the concentration of the secretory component (SC) of p-immunoglobulin A in subcellular fractions of crypt cells in 35-day-old rats made iron deficient from birth and in controls. The aim of the study is to investigate the biochemical basis for the decreased activity of brush-border disaccharidases observed in growing animals with chronic iron deficiency. In rats made iron deficient, the specific (per unit protein) and the total (per total intestinal length) activities of sucrase, lactase, maltase, aminopeptidase, and diamine oxidase were decreased from -17 to -66% compared with the activities measured in the controls. The lower activity of sucrase in the brush-border membrane of the iron-deficient rats was associated with much slower enzyme synthesis rate than in control animals. Km of sucrase was identical in both iron-deficient rats and controls, but the maximum velocity of enzyme reaction was reduced proportionally to the enzymatic activity, indicating a lesser amount of enzyme rather than an inactivation. Electron microscopy of epithelial villous cells from iron-deficient rats revealed a marked rarefaction of secretory granules (transport vesicles) without apparent change in the morphology of the brush-border membrane or of cellular organelles. In villus and crypt cells isolated from the jejunum of iron-deficient rats, SC concentration was reduced to a level about half that of the controls. When SC concentration was measured in subcellular fractions of crypt cells, SC content in each fraction was two to three times lower in iron-deprived rats than in controls without evidence of accumulation of the protein at a given subcellular level.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of Sucrose and Starch on the Development of Anemia in Copper- and Iron-Deficient Rats

The purpose of this investigation was to compare the effects of sucrose and starch on the development of copper and iron deficiency. Male Sprague-Dawley rats (n = 48) were fed one of eight diets in a 2 × 2 × 2 factorial design for 24 d. Two levels of copper (deficient, 0.7 µg/g, or adequate, 8.3 µg/g) and iron (deficient, 8.3 µg/g, or adequate, 50 µg/g), and two types of carbohydrate (sucrose or starch, 62% of the diet) were fed. Copper-deficient rats had significantly lower hematocrit, hemoglobin and tibia iron levels and depressed copper and iron absorption when fed sucrose instead of starch. The apparent absorption of copper, but not iron, was significantly lower when rats deficient in both copper and iron were fed sucrose rather than starch. Iron-deficient rats fed sucrose apparently absorbed significantly more iron than those fed starch; however, sucrose did not significantly improve hematocrit and hemoglobin levels. The metabolism of copper and iron by rats fed diets adequate in these nutrients was not affected by the type of dietary carbohydrate. These data indicate that the type of dietary carbohydrate alters both copper and iron metabolism, particularly in copper-deficient rats.

Independence of in vitro iron absorption from mucosal transferrin content in rat jejunal and ileal segments.

Isolated non blood-perfused intestinal segments from normal and iron-deficient rats were used in vitro. A modification of the luminal perfusion method according to Fisher and Parsons allowed the comparison of iron and transferrin quantities in the serosal fluid at 15 min intervals. Iron transfer in jejunal and ileal segments was directly proportional to the luminal iron concentration within a dose range of 1 to 100 mumol/l, did not show saturation characteristics and was linear over time. Jejunal segments from iron-deficient rats transferred about twice as much iron as the jejunal controls. In ileal segments there was no difference in iron transfer between iron-deficient and control rats; in both cases transfer amounted to approx. 10% of jejunal controls. An exponential correlation was found, when the decreasing transferrin content of the tissue was plotted against the cumulative water transport. Transferrin and albumin release from jejunal and ileal segments into the absorbate cumulated asymptotically, which is typical for wash-out phenomena. As iron transfer cumulated linearly while transferrin release cumulated in an asymptotic manner, the capacity of transferrin to bind iron ions is exceeded roughly 100 times by molar equivalents of iron in the last absorbate fractions. Independence of iron transfer from mucosal transferrin quantities is concluded. As the molar transferrin/albumin ratios do not show significant differences between plasma and the sequence of absorbate samples, a wash-out from the gut's interstitial space is assumed, which makes plasma the most likely origin of transferrin in the mucosa.

Postweaning Carnitine Supplementation of Iron-Deficient Rats

Severe iron deficiency in the suckling and weanling rat is associated with lipid accumulation in serum and liver, impaired ketogenesis in the suckling pup and low levels of carnitine in some tissues. Carnitine has been effective in reducing high triacylglycerol levels in humans and rats. This study examined tissue triacylglycerol concentrations of iron-deficient rats supplemented with carnitine or iron. Iron-adequate (C) and iron-deficient (D) pups were weaned to diets containing 38 ppm Fe (c) or 6 ppm Fe (d) with or without 0.2% DL-carnitine (Carn) resulting in six experimental treatments: CcCarn, DdCarn, Cc, Cd, Dc, Dd. Males received the diets for 2 wk and female littermates for 4. After 2 and 4 wk, carnitine supplementation significantly increased carnitine content in liver, heart and skeletal muscle by 30–60% in rats from control and Fe-deficient dams. Carnitine treatment significantly lowered the triacylglycerol level in liver of 49-d-old Fe-deficient females, but did not affect other tissues at either time point compared to other dietary treatments. Fe supplementation did not increase carnitine content in tissues, but did reduce triacylglycerol levels in liver by 4 wk and in skeletal muscle at both time points. Possible mechanisms by which iron and carnitine may lower lipids are discussed.