Effect of Latent Iron Deficiency on 5-Hydroxytryptamine Metabolism in Rat Brain

Eight weeks of latent iron deficiency in weaned rats maintained on an experimental low iron content diet (18-20 mg/kg) did not significantly alter the packed cell volume and hemoglobin concentration; however, the hepatic and brain nonheme iron contents decreased by 66% and 21% (p less than 0.001), respectively. The tryptophan concentration decreased by 31% and 34% in liver and brain, respectively, in rats on experimental diet (p less than 0.01). The brain 5-hydroxytryptamine and 5-hydroxyindoleacetic acid contents were reduced by 21% and 23% (p less than 0.01 and p less than 0.02), respectively. However, in the brain, weight, protein, DNA, and the activities of monoamine oxidase, aldehyde dehydrogenase, and liver tryptophan oxygenase were found to remain unaltered. When rehabilitated with a diet containing 390 mg/kg iron, rats previously maintained on the experimental diet for 2 weeks showed partial recovery in tryptophan levels both in liver and brain. However, brain 5-hydroxytryptamine and 5-hydroxyindoleacetic acid levels remained unaltered. The hepatic iron content improved without any change in brain iron content. The latent iron deficiency produced significant alterations in the metabolism of 5-hydroxytryptamine and brain iron content that could not be recovered 2 weeks after the iron rehabilitation.     

Effect of latent iron deficiency on metal levels of rat brain regions

Seven different metals (iron, copper, zinc, calcium, manganese, lead, and cadmium) were studied in eight different brain regions (cerebral cortex, cerebellum, corpus striatum, hypothalamus, hippocampus, midbrain, medulla oblongata, and pons) of weaned rats (21-d-old) maintained on an iron-deficient (18-20 mg iron/kg) diet for 8 wk. Iron was found to decrease in all the brain regions, except medulla oblongata and pons, in comparison to their respective levels in control rats, receiving an iron-sufficient (390 mg iron/kg) diet. Brain regions showed different susceptibility toward iron deficiency-induced alterations in the levels of various metals, such as zinc, was found to increase in hippocampus (19%, p less than 0.05) and midbrain (16%, p less than 0.05), copper in cerebral cortex (18%, p less than 0.05) and corpus striatum (16% p less than 0.05), calcium in corpus striatum (22%, p less than 0.01) and hypothalamus (17%, p less than 0.02), and manganese in hypothalamus (18%, p less than 0.05) only. Toxic metals lead and cadmium also increased in cerebellum (19%, p less than 0.05) and hippocampus (17%, p less than 0.05) regions, respectively. Apart from these changes, liver (64%, p less than 0.001) and brain (19%, p less than 0.01) nonheme iron contents were found to decrease significantly, but body, liver, and brain weights, packed cell volume, and hemoglobin content remained unaltered in these experimental rats. Rehabilitation of iron-deficient rats with an iron-sufficient diet for 2 wk recovered the values of zinc in both the hippocampus and mid-brain regions and calcium in the hypothalamus region only. Liver nonheme iron improved significantly; however, no remarkable effect was noticed in brain nonheme iron following rehabilitation. It may be concluded that latent iron deficiency produced alterations in various metal levels in different brain regions, and corpus striatum was found to be the most vulnerable region for such changes. It is also evident that brain regions were resistant for any recovery in their altered metallic levels in response to rehabilitation for 2 wk.     

Iron bioavailability from fortified fluid milk and petit suisse cheese determined by the prophylactic-preventive method

In this research, we measure the iron bioavailability of micronized ferric orthophosphate when it is used to fortify low-fat fluid milk enriched with calcium and petit suisse cheese using the prophylactic-preventive method in rats. Four groups of male weaned rats received a basal diet (control diet; 6.5 ppm Fe), a reference standard diet (SO₄Fe; 18.2 ppm Fe), a basal diet using iron-fortified fluid milk as the iron source (milk diet; Fe ppm 17.9), and a basal diet using iron-fortified petit suisse cheese as the iron source (cheese diet; 18.0 ppm Fe) for 22 d. The iron bioavailability of the different sources was calculated as the ratio between the mass of iron incorporated into hemoglobin during the experiment and the total iron intake per animal. The relative biological values with regard to the reference standard (RBV%) were 61% and 69% for the milk and cheese diet, respectively. These results show that according to this method, the iron bioavailability in both fortified foods can be considered as medium bioavailability rates.     

Iron overload,measured as serum ferritin,increases brain damage induced by focal ischemia and early reperfusion

High levels of iron, measured as serum ferritin, are associated to a worse outcome after stroke. However, it is not known whether ischemic damage might increase ferritin levels as an acute phase protein or whether iron overload affects stroke outcome. The objectives are to study the effect of stroke on serum ferritin and the contribution of iron overload to ischemic damage.Swiss mice were fed with a standard diet or with a diet supplemented with 2.5% carbonyl iron to produce iron overload. Mice were submitted to permanent (by ligature and by in situ thromboembolic models) or transient focal ischemia (by ligature for 1 or 3 h).Treatment with iron diet produced an increase in the basal levels of ferritin in all the groups. However, serum ferritin did not change after ischemia. Animals submitted to permanent ischemia had the same infarct volume in the groups studied. However, in mice submitted to transient ischemia followed by early (1 h) but not late reperfusion (3 h), iron overload increased ischemic damage and haemorrhagic transformation.Iron worsens ischemic damage induced by transient ischemia and early reperfusion. In addition, ferritin is a good indicator of body iron levels but not an acute phase protein after ischemia.     

Iron bioavailability from fortified petit suisse cheese determined by the prophylactic-preventive method

In this research, we measured the iron bioavailability of ferrous gluconate stabilized with glycine (SFG) when it is used to fortify petit suisse cheese using the prophylactic-preventive method in rats. Three groups of male, weaned rats received a basal diet (control diet; 5.2 ppm Fe), a reference standard diet (SO₄Fe; 9.2 ppm Fe), and a basal diet using iron-fortified petit suisse cheese as the iron source (cheese diet; 8.8 ppm Fe) for 22d. The iron bioavailability was calculated as the ratio between the mass of iron incorporated into hemoglobin and the total iron intake per animal during the treatment. These values (BioFe) were 68% and 72% for SFG and ferrous sulfate, respectively. The value of the Relative Biological Value (RBV) was 95% for SFG in petit suisse cheese. These results show that according to this method, the iron bioavailability from industrial fortified petit suisse cheese can be considered as a high bioavailability rate.     

Hemochromatosis: Evaluation of the dietary iron model and regulation of hepcidin

Our knowledge of iron homeostasis has increased steadily over the last two decades; much of this has been made possible through the study of animal models of iron-related disease. Analysis of transgenic mice with deletions or perturbations in genes known to be involved in systemic or local regulation of iron metabolism has been particularly informative. The effect of these genes on iron accumulation and hepcidin regulation is traditionally compared with wildtype mice fed a high iron diet, most often a 2% carbonyl iron diet. Recent studies have indicated that a very high iron diet could be detrimental to the health of the mice and could potentially affect homeostasis of other metals, for example zinc and copper. We analyzed mice fed a diet containing either 0.25%, 0.5%, 1% or 2% carbonyl iron for two weeks and compared them with mice on a control diet. Our results indicate that a 0.25% carbonyl iron diet is sufficient to induce maximal hepatic hepcidin response. Importantly these results also demonstrate that in a chronic setting of iron administration, the amount of excess hepatic iron may not further influence hepcidin regulation and that expression of hepcidin plateaus at lower hepatic iron levels. These studies provide further insights into the regulation of this important hormone.     

Vitamin A deficiency modulates iron metabolism via ineffective erythropoiesis

Vitamin A modulates inflammatory status, iron metabolism and erythropoiesis. Given that these factors modulate the expression of the hormone hepcidin (Hamp), we investigated the effect of vitamin A deficiency on molecular biomarkers of iron metabolism, the inflammatory response and the erythropoietic system. Five groups of male Wistar rats were treated: control (AIN-93G), the vitamin A-deficient (VAD) diet, the iron-deficient (FeD) diet, the vitamin A- and iron-deficient (VAFeD) diet or the diet with 12 mg atRA/kg diet replacing all-trans-retinyl palmitate by all-trans retinoic acid (atRA). Vitamin A deficiency reduced serum iron and transferrin saturation levels, increased spleen iron concentrations, reduced hepatic Hamp and kidney erythropoietin messenger RNA (mRNA) levels and up-regulated hepatic and spleen heme oxygenase-1 gene expression while reducing the liver HO-1 specific activity compared with the control. The FeD and VAFeD rats exhibited lower levels of serum iron and transferrin saturation, lower iron concentrations in tissues and lower hepatic Hamp mRNA levels compared with the control. The treatment with atRA resulted in lower serum iron and transferrin concentrations, an increased iron concentration in the liver, a decreased iron concentration in the spleen and in the gut, and decreased hepatic Hamp mRNA levels. In summary, these findings suggest that vitamin A deficiency leads to ineffective erythropoiesis by the down-regulation of renal erythropoietin expression in the kidney, resulting in erythrocyte malformation and the consequent accumulation of the heme group in the spleen. Vitamin A deficiency indirectly modulates systemic iron homeostasis by enhancing erythrophagocytosis of undifferentiated erythrocytes.     

Plasma iron is associated with lipid peroxidation in an elderly population

Epidemiological evidence has raised concern that a moderate elevation in body iron stores may increase oxidative stress and risk of heart disease. We examined the cross-sectional association between plasma iron and factors that could affect its levels (antioxidant enzymes, diet), with the concentration of plasma malondialdehyde (MDA) as a marker of lipid peroxidation. Participants were 162 non-smoking institutionalised elderly. Our results show that those in the highest tertile of plasma iron were at least twice as likely to have higher plasma MDA levels. Among the factors affecting plasma iron levels, we found that the upper tertile of erythrocyte-superoxide dismutase (E-SOD) was inversely associated with higher plasma iron, and potato intake explained a sizeable proportion of the variation in plasma iron levels. In addition to potatoes, eggs, wine, fruit in men and green vegetables in women showed a positive association with plasma iron levels. Only potatoes in both sexes, wine in men and eggs in women had an independent effect on plasma MDA. Potatoes, wine, plasma lycopene and plasma iron accounted for 43% of the variability in plasma MDA for males, and E-SOD, potatoes, eggs, plasma lycopene and plasma iron explained 45% for women. A longitudinal study should confirm, whether these MDA levels are related to morbidity and mortality.     

Effect of Dietary Iron Loading on Recognition Memory in Growing Rats

While nutritional and neurobehavioral problems are associated with both iron deficiency during growth and overload in the elderly, the effect of iron loading in growing ages on neurobehavioral performance has not been fully explored. To characterize the role of dietary iron loading in memory function in the young, weanling rats were fed iron-loading diet (10,000 mg iron/kg diet) or iron-adequate control diet (50 mg/kg) for one month, during which a battery of behavioral tests were conducted. Iron-loaded rats displayed elevated non-heme iron levels in serum and liver, indicating a condition of systemic iron overload. In the brain, non-heme iron was elevated in the prefrontal cortex of iron-loaded rats compared with controls, whereas there was no difference in iron content in other brain regions between the two diet groups. While iron loading did not alter motor coordination or anxiety-like behavior, iron-loaded rats exhibited a better recognition memory, as represented by an increased novel object recognition index (22% increase from the reference value) than control rats (12% increase; P=0.047). Western blot analysis showed an up-regulation of dopamine receptor 1 in the prefrontal cortex from iron-loaded rats (142% increase; P=0.002). Furthermore, levels of glutamate receptors (both NMDA and AMPA) and nicotinic acetylcholine receptor (nAChR) were significantly elevated in the prefrontal cortex of iron-loaded rats (62% increase in NR1; 70% increase in Glu1A; 115% increase in nAChR). Dietary iron loading also increased the expression of NMDA receptors and nAChR in the hippocampus. These results support the idea that iron is essential for learning and memory and further reveal that iron supplementation during developmental and rapidly growing periods of life improves memory performance. Our investigation also demonstrates that both cholinergic and glutamatergic neurotransmission pathways are regulated by dietary iron and provides a molecular basis for the role of iron loading in improved memory.     

Does nitric oxide contribute to iron-dependent brain injury after experimental cerebral ischaemia?

Experimental and clinical data suggest that iron has a key role in cerebral ischaemia. We measure infarct volume and analyse the nitric oxide responses to brain injury in rat stroke model after increased oral iron intake. Permanent middle cerebral artery occlusion (MCAO) was performed in a group of 20 male Wistar rats, 10 of which were fed with a control diet and 10 of which were fed with iron-enriched diet containing 2.5% carbonyl iron for 9 weeks. L-arginine and nitric oxide metabolites were determined in blood samples before and at 2, 6, 8 and 48 h after MCAO. Infarct volume, thiobarbituric acid reaction substances (TBARS) and tissue iron were measured at 48 h. Infarct volume was 66% greater in the iron-fed rats than in the control group. Iron-fed animals showed significantly higher levels of TBARS. Liver iron stores (3500 +/- 199 vs 352 +/- 28 microg Fe/g, p<0.0001) but not brain iron stores (131 +/- 11 vs 139 +/- 8 microg Fe/g, p=0.617), were significantly higher in the iron-fed group. L-arginine levels were slightly lower in iron-fed rats and decreased significantly in both groups at 6 and 8 hours after MCAO. The levels of the stable end products of NOS (NOx = nitrite + nitrate) were significantly higher in iron-fed rats before MCAO (16.2 +/- 2.2 vs. 9.6 +/- 0.8 micromol x L(-1), p<0.05), with a further increase during the six first hours after MCAO in both groups. These results suggest that the iron overload that increases both superoxide and nitric oxide production leads to peroxynitrite formation, thus enhancing brain damage.     

Iron metabolism in Trypanosoma lewisi infection: serum iron and serum iron-binding capacity.

Progressive changes in iron levels, total iron binding capacity and hematocrit values in sera of rats infected with Trypanosoma lewisi are described. The host dietary group were: (1) complete or full complement; (2) iron-deficient, and (3) pair-fed or calorically restricted. The hematocrit values of T. lewisi-infected rats given the various diets were not significantly different from those of the controls. The decrease in total iron binding capacity (TIBC) of rats inoculated with T. lewisi and fed complete and pair-fed diets ranged up to 15% over uninfected controls. TIBC levels in rats fed an iron-deficient diet and inoculated with T. lewisi ranged up to 32% over uninfected controls. TIBC levels of deficient infected rats were significantly different from the controls from day 90 to infection to the end of the observation period. Serum iron (SI) values of non-infected rats regardless of dietary regimen showed significantly higher values than T. lewisi-infected animals between days 95 and 120. The average SI value, for this period, in adequately fed control rats was 204 +/- 7 microgram/100 ml as compared to 172 +/- 5 microgram/100 for trypanosome-infected rats. SI levels of rats on a pair-fed diet and infected with T. lewisi decreased to 17% over uninfected controls. SI levels of animals on an iron-deficient diet and infected with T. lewisi decreased up to 76% over uninfected controls.     

The effect of different dietary iron levels on growth and hepatic iron concentration in juvenile gibel carp (Carassius auratus gibelio)

An 83-day growth trial was conducted using a flow-through system to examine the effects of different dietary iron levels on growth and hepatic iron concentration in juvenile gibel carp ( Carassius auratus gibelio ). Six purified diets supplemented with different levels of iron (0, 10, 30, 60, 100 and 200 mg kg⁻¹) (as ferrous sulfate) were fed to triplicate groups of fish (initial weight 2.12 ± 0.00 g per fish). The results showed that the addition of iron to the basal diet did not significantly affect the specific growth rate (SGR), feed efficiency (FE), survival, red blood cell amount (RBC), hemoglobin content (Hb), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) or mean corpuscular hemoglobin concentration (MCHC). Hepatic iron concentration and hematocrit (Hct) were significantly influenced by dietary iron level (P < 0.05). On the basis of the iron concentration for the maintenance of optimum hepatic iron concentration and Hct, it was concluded that the dietary iron concentration of juvenile gibel carp should be not less than 202 mg Fe kg⁻¹ diet.     

Effect of lithium on hepatic and serum elemental status under different dietary protein regimens

Lithium carbonate at the dose level of 1.1 g/kg was administered in diet to normal (18% protein), low-protein-(LP; 8%) and highprotein (HP; 30% diet)-fed rats for a period of 1 mo. The LP diet resulted in a significant decrease in the hepatic levels of zinc, iron, copper, manganese, calcium, and serum levels of calcium and sodium. The HP diet caused a marked decrease in copper and calcium levels in liver, but an increase in potassium levels in serum was observed. Lithium treatment to normal rats led to a significant reduction in the hepatic contents of zinc, copper, potassium, calcium, and serum contents of potassium and sodium, whereas an elevation in serum contents of calcium was noticed. Administration of lithium to protein-deficient rats increased the hepatic concentration of manganese and serum concentration of calcium and the levels almost reached the normal limits. On the other hand, there was a marked depression in potassium contents in the serum of LP-as well as HP-fed rats following lithium treatment when compared to LP and HP groups, respectively.     

Serum indices of body stores of iron in northern fur seals (Callorhinus ursinus) and their relationship to hemochromatosis

Iron storage disease (hemochromatosis) has been reported in many species of both captive and free‐ranging animals. In this study we examined the relationship between this disease and concentrations of iron analytes in aquarium‐held northern fur seals (Callorhinus ursinus). Sera were analyzed for iron, total iron‐binding capacity (TIBC), ferritin, ceruloplasmin, and haptoglobin concentrations in a retrospective study that included samples taken over a 14‐year period. The animals ranged in age from <1 year to an estimated 23 years. Serum ferritin was measured using an enzyme‐linked immunosorbent assay (ELISA) for canine sera. The results from this assay are the first reported for any pinniped. Serum iron concentrations in presumed healthy animals ranged from 37 to 196 µg/dl, and TIBC ranged from 136 to 484 µg/dl. The transferrin saturation percentage differed significantly between male (41%) and female (63%) adult fur seals, as did the ferritin levels (54 ng/ml for males vs. 500 ng/ml for females). There was a trend toward increased serum ferritin and percent transferrin saturation with age, especially in females. The data also showed a relationship between serum iron and transferrin saturation among eight mother–pup pairs, which suggests that pups may develop increased iron levels due to placental transfer of iron and/or transfer of iron through the milk from iron‐overloaded females. Diet was considered as a factor in the development of hemochromatosis in at least three geriatric female northern fur seals, and their diets were analyzed for iron concentrations. On the basis of these results, the diets were altered by replacing a portion of the high‐iron‐content fish (herring) with a lower‐iron‐content item (squid), and discontinuing iron and vitamin C supplementation (via a multivitamin tablet). Sera were analyzed before, and 1 and 4 years after the dietary changes were implemented. Paired t‐tests showed no significant changes in the iron analytes from pre‐ to post‐diet‐change samples, which indicates that it may be too late to affect iron levels by diet alone in older animals with a chronic history of elevated iron levels. Zoo Biol 23:205–218, 2004. © 2004 Wiley‐Liss, Inc.     

Oxidative stress and antioxidant status in mouse kidney: effects of dietary lipid and vitamin E plus iron

The purpose of this study was to determine the effects of dietary fat, vitamin E, and iron on oxidative damage and antioxidant status in kidneys of mice. Sixty 1-month-old male Swiss-Webster mice were fed a basal vitamin E-deficient diet that contained either 8% fish oil + 2% corn oil or 10% lard with or without 1 g all-rac-alpha-tocopherol acetate or 0.74 g ferric citrate per kilogram of diet for 4 weeks. Significantly (P < 0.05) higher levels of lipid peroxidation products, thiobarbituric acid reactants (TBAR), and conjugated dienes were found in the kidneys of mice fed with fish oil compared with mice fed lard irrespective of vitamin E status. Mice maintained on a vitamin E-deficient diet had significantly higher renal levels of TBAR, but not conjugated dienes, than the supplemented group. Fish oil fed mice receiving vitamin E supplementation had lower levels of alpha-tocopherol than did mice in the lard fed group. Significantly higher levels of ascorbic acid were also found in the kidneys of mice fed with fish oil than were found in mice fed lard. The levels of protein carbonyls and glutathione (GSH), and activities of catalase, superoxide dismutase, selenium (Se)-GSH peroxidase, and non-Se-GSH peroxidase were not significantly altered by dietary fat or vitamin E. Dietary iron had no significant effect on any of the oxidative stress and antioxidant indices measured. The results obtained provide experimental evidence for the pro-oxidant effect of high fish oil intake in mouse kidney and suggest that dietary lipids play a key role in determining cellular susceptibility to oxidative stress.     

Impaired insulin secretion and decreased expression of the nutritionally responsive ribosomal kinase protein S6K-1 in pancreatic islets from malnourished rats

Low protein diet has been shown to affect the levels and activities of several enzymes from pancreatic islets. To further extend the knowledge on how malnutrition affects insulin secretion pathway, we investigated in this work the insulin release induced by glucose or leucine, an insulin secretagogue, and the expression of insulin receptor (IR), insulin receptor substrate 1 (IRS1), phosphatidylinositol 3-kinase (PI3K), and p70S6K1 (S6K-1) proteins from pancreatic islets of rats fed a normal (17%; NP) or a low (6%; LP) protein diet for 8 weeks. Isolated islets were incubated for 1 h in Krebs-bicarbonate solution containing 16.7 mmol/L of glucose, or 2.8 mmol/L of glucose in the presence or absence of 20 mmol/L of leucine. Glucose- and leucine-induced insulin secretions were higher in NP than in LP islets. Western blotting analysis showed an increase in the expression of IR and PI3K protein levels whereas IRS1 and S6K-1 protein expression were lower in LP compared to NP islets. In addition, S6K-1 mRNA expression was also reduced in islets from LP rats. Our data indicate that a low protein diet modulates the levels of several proteins involved in the insulin secretion pathway. Particularly, the decrease in S6K-1 expression might be an important factor affecting either glucose- or leucine-induced insulin secretion.     

Changes of Linoleic,Arachidonic and Eicosapentaenoic Acids in Rat Platelet,Aorta and Plasma Lipids after Changing from a Sardine Oil Diet to a Corn Oil Diet

When rats were fed on a diet containing sardine oil (SO) for 10 days, 20:5 (eicosapentaenoic acid) was incorporated into the platelet, aorta and plasma lipids. The 20:5/20:4 (arachidonic acid) ratios of phospholipids were higher in platelets than in aortas. When the diet was changed from SO to corn oil (CO), the levels of 20:5 in the plasma lipids and platelet phospholipids decreased with an increase of 20:4. Although the 20:5 levels of phospholipids in the aortas decreased, 20:4 did not increase during feeding with the CO diet for 4 days. The 20:5/20:4 ratios of the phosphatidylcholine in platelets and aortas rapidly changed from 5.0 and 1.1 to 2.4 and 0.6, respectively. The linoleic acid level of phosphatidylcholine in platelets and aortas increased rapidly by changing from the SO diet to the CO diet, but this fatty acid was not incorporated into phosphatidylinositol of the platelets and aortas.     

Dietary intrinsic phytate protects colon from lipid peroxidation in pigs with a moderately high dietary iron intake.

High iron consumption has been proposed to relate to an increase in the risk of colon cancer, whereas high levels of supplemental sodium phytate effectively reduce iron-induced oxidative injury and reverse iron-dependent augmentation of colorectal tumorigenesis. However, the protective role of intrinsic dietary phytate has not been determined. In this study, we examined the impact of removing phytate present in a corn-soy diet by supplemental microbial phytase on susceptibility of pigs to the oxidative stress caused by a moderately high dietary iron intake. Thirty-two weanling pigs were fed the corn-soy diets containing two levels of iron (as ferrous sulfate, 80 or 750 mg/kg diet) and microbial phytase (as Natuphos, BASF, Mt. Olive, NJ, 0 or 1200 units/kg). Pigs fed the phytase-supplemented diets did not receive any inorganic phosphorus to ensure adequate degradation of phytate. After 4 months of feeding, liver, colon, and colon mucosal scrapings were collected from four pigs in each of the four dietary groups. Colonic lipid peroxidation, measured as thiobarbituric acid reacting substances (TBARS), was increased by both the high iron (P< 0.0008) and phytase (P< 0.04) supplementation. Both TBARS and F2-isoprostanes, an in vivo marker of lipid peroxidation, in colonic mucosa were affected by dietary levels of iron (P< 0.03). Mean hepatic TBARS in pigs fed the phytase-supplemented, high iron diet was 43%-65% higher than that of other groups although the differences were nonsignificant. Moderately high dietary iron induced hepatic glutathione peroxidase activity (P= 0.06) and protein expression, but decreased catalase (P< 0.05) in the colonic mucosa. In conclusion, intrinsic phytate in corn and soy was protective against lipid peroxidation in the colon associated with a moderately high level of dietary iron.