Phagocytosis and lysozyme activity in granulocytes from iron-deficient rat dams and pups

The effect of dietary iron deficiency during gestation and lactation on phagocyte function in rat dams and pups was investigated. Pregnant rats were fed ad libitum diets containing 7, 10 or 250 ppm iron throughout gestation and until day 17 of lactation when dams and their litters (7 pups) were sacrificed. Phagocytosis was measured by the nitroblue tetrazolium (NBT) dye reduction test. Lysozyme activity was measured in granulocytes separated from peripheral blood. A significant increase in percent phagocytes in whole blood in the 7 ppm iron pups was observed. Total white blood cell (WBC) counts were similar among groups. The 7 ppm iron pups had markedly decreased NBT reduction (per 10⁸ phagocytes). On a per ml of blood basis, there were no differences in NBT reduction among groups. Plasma activities of the two granulocyte enzymes measured, lysozyme and peroxidase, were significantly increased in 7 ppm iron pups. It was shown that this increase in plasma lysozyme activity was not due to an increased concentration of lysozyme in granulocytes.     

Early postnatal iron repletion overcomes lasting effects of gestational iron deficiency in rats

Iron deficiency anemia in early childhood causes developmental delays and, very likely, irreversible alterations in neurological functioning. One primary goal for the present study was to determine whether the effects of late gestational iron deficiency on brain monoamine metabolism, iron content, and behavioral phenotypes could be repaired with iron intervention in early lactation. Young pregnant rats were provided iron-deficient or control diets from mid-gestation (G15). At postnatal d 4 (P4), pups from iron-deficient dams were out-fostered either to other ID dams or control dams while pups of control dams were similarly fostered to other control dams. Dietary treatments continued to adulthood (P65) when brain iron and regional monoamines were evaluated. P4 iron repletion normalized body iron status, brain iron concentrations, monoamine concentrations, and monoamine transporter and receptor densities in most brain regions. Dopamine transporter densities in caudate and substantia nigra were lower in ID rats but were normalized with iron repletion. Serotonin transporter levels in most brain regions and open-field exploration were also normalized with iron repletion. The success of this approach of early postnatal iron intervention following iron deficiency in utero contrasts to a relative lack of success when the intervention is performed at weaning. These data suggest that a window of opportunity exists for reversing the detrimental effects of iron deficiency in utero in rats and provides strong support of intervention approaches in humans with iron deficiency during pregnancy.     

Decreased antibody formation in iron-deficient rat pups--effect of iron repletion

Rats were fat diets containing 6, 12, or 250 ppm iron throughout gestation and lactation. On day 17, pups immunized with sRBC were used to determine antibody synthesis by the Jerne plaque assay. In both iron-deficient groups, antibody formation was decreased by at least 50% compared to controls. For 3 weeks beginning on day 21, iron-deficient pups were fed either a control diet (35 ppm iron) or the same iron-deficient diet as fed to the dam. IgG and IgM formation was only slightly improved in repleted rats and remained significantly below that of rats fed the control diet throughout the experiment. In contrast, 250 ppm iron pups fed an iron-deficient diet postweaning had significantly decreased IgG and IgM production compared to littermates fed a control diet postweaning. Maternal iron deficiency during the critical pre- and postnatal growth periods may result in long-term impairment of humoral immunity that is not corrected by dietary iron repletion after weaning.     

A postweaning iron-adequate diet following neonatal iron deficiency affects iron homeostasis and growth in young rats

Iron deficiency is among the most prevalent of nutrient-related diseases worldwide, but the long-term consequences of maternal and neonatal iron deficiency on offspring are not well characterized. We investigated the effects of a postweaning iron-adequate diet following neonatal iron deficiency on the expression of genes involved in iron acquisition and homeostasis. Pregnant rats were fed an iron-adequate diet (0.08 g iron/kg diet) until gestational d 15, at which time they were divided into 2 groups: 1) a control group fed an iron-adequate diet, and 2) an iron-deficient group fed an iron-deficient diet (0.005 g iron/kg diet) through postnatal d (P) 23 (weaning). After weaning, pups from both dietary treatment groups were fed an iron-adequate diet until adulthood (P75). Rat pups that were iron deficient during the neonatal period (IDIA) had reduced weight gain and hemoglobin concentrations and decreased levels of serum, liver, and spleen iron on P75 compared with rats that were iron sufficient throughout early life (IA). IDIA rats developed erythrocytosis during postweaning development. Further, hepatic expression of hepcidin in IDIA rats was 1.4-fold greater than in IA rats, which paralleled an upregulation of IL-1 expression in the serum. Our data suggest that an iron-adequate diet following neonatal iron deficiency induced an inflammatory milieu that affected iron homeostasis and early growth and development.     

Impaired milk folate secretion is not corrected by supplemental folate during iron deficiency in rats

Decreased milk folate secretion in iron-deficient rat dams contributes to the impairment of folate metabolism in nursing pups. The present study was designed to assess whether impaired milk folate secretion secondary to iron deficiency is due to a decrease in the supply of folate to the mammary gland or to an inability of the mammary gland to effectively use folate. Rats were fed diets containing 0.5, 2.0 or 7.0 mg folate/kg and 8 (-Fe) or 250 (+Fe) mg Fe/kg throughout gestation and until d 17 of lactation. Regardless of dietary Fe content, maternal plasma, red blood cell, liver and kidney folate concentrations correlated with dietary folate content (r = 0.75-0.85, p less than 0.0001). With the exception of plasma folate level, which was 46% lower for -Fe than +Fe dams fed 0.5 mg folate/kg, no other differences in indices of folate status were noted between +Fe and -Fe dams. Dietary folate content had a direct impact on milk folate content in +Fe dams but not in -Fe dams. Mammary tissue methionine synthase and folylpolyglutamate synthetase activities were not depressed in Fe deficiency; rather, mean activities were elevated among -Fe dams fed 0.5 mg folate/kg. In conclusion, the reduction in milk folate secretion during Fe deficiency is not due to a decrease in the amount of folate supplied to the mammary gland; rather, the defect causing this reduction is specific to the mammary gland.     

Iron deficiency hyperlipidemia in 18-day-old rat pups: effects of milk lipids, lipoprotein lipase, and triglyceride synthesis

Three levels of iron (5, 29, 307 ppm iron) were fed to rats from conception through the 18th day of lactation. Dams in the 5 ppm iron group and pups in the 5 and 29 ppm iron groups developed anemia characterized by lower hemoglobin and hematocrit values than control animals. Liver and spleen levels of iron in dams and pups in the 5 and 29 ppm iron groups were lower than in the 307 ppm iron groups. Milk iron was lower in the 5 ppm iron group than in the 29 and 307 ppm iron groups. Pups in the 5 ppm iron group had hyperlipidemia characterized by elevated serum triglycerides, cholesterol, and phospholipids. Milk lipids and post-heparin plasma lipoprotein lipase levels in pups did not differ among experimental groups. Triglyceride and CO2 production from [U-14C]glucose were significantly greater in the iron-deficient pups than in control pups. Hyperlipidemia in 18-day-old iron-deficient rat pups appears to be related to increased endogenous production of triglycerides.     

Depressed folate incorporation into milk secondary to iron deficiency in the rat

The present study was designed to determine whether reduced folate incorporation into milk can account for folate depletion of iron-deficient suckling rats. Dams were fed diets containing 2 mg/kg folate and either 8, 12 or 250 mg/kg iron throughout gestation and lactation to produce severely iron-deficient, moderately iron-depleted and iron-sufficient states in 17-d-old pups (n = 15 litters/group). On d 17 of lactation, dams were separated from litters and given intraperitoneal injections of [3',5',7,9-3H]pteroylmonoglutamic acid ([3H]PteGlu) or physiological saline. Mean [3H]PteGlu incorporation into milk of severely iron-deficient dams was 67% of that in iron-sufficient controls, while "total" and "free" milk folate activities were 54 and 61%, respectively. Values for milk [3H]PteGlu incorporation and folate activities were intermediate in moderately iron-depleted dams. Pup red blood cell folate activity was positively correlated with both free (r = 0.43, P = 0.004) and total (r = 0.37, P = 0.015) milk folate activities. Mean plasma folate activities of severely and moderately iron-deficient pups were 68 and 86% of control values, respectively. Results show that in both mild and severe iron deficiency, reduced folate secretion into milk is at least partially responsible for impaired folate status of suckling pups.     

Iron deficiency impairs protein synthesis in immune tissues of rat pups

To determine if decreased protein synthesis is a factor in reduced immunocompetence of iron deficiency, RNA, DNA and in vitro protein synthesis were measured. Rats were fed diets containing 6 (severe anemia), 11 (moderate anemia) or 250 (iron sufficient) mg iron/kg diet throughout gestation and lactation. On d 2 of lactation, litters were adjusted to contain six pups. On d 12 of lactation, two pups from each litter were immunized with sheep red blood cells (SRBC) and on d 17, tissues were removed for the determination of protein synthesis and evaluation of RNA and DNA contents. In the moderately iron-deficient pups, protein synthesis was lower (30%) in spleen than that in iron-sufficient pups. Protein synthesis in liver and thymus was not changed by moderate iron deficiency. In spleen, liver and thymus, protein synthesis in severely iron-deficient pups was less than half that of iron-sufficient pups. Protein synthesis in the spleen of the moderately iron-deficient group was higher after immunization with SRBC than in iron-sufficient controls, whereas the severely iron-deficient pups failed to respond. Impaired protein synthesis may be the mechanism responsible for compromised ability to produce antibody in iron deficiency.     

Copper and iron deficiencies: effects on serum lipids and tissue minerals

To study the interrelationship between iron and copper on serum lipid concentrations, four diets were fed to growing rats: iron and copper deficient, copper-deficient, iron-deficient, iron and copper adequate. After 18 weeks, concentrations of iron and copper in organs and lipids in sera were determined. Iron deficiency alone or combined with copper deficiency resulted in reduced body weights, hemoglobin concentrations, hematocrits, and iron concentrations in liver, spleen, and heart. Hepatic copper was elevated 8-fold in iron deficiency. Copper deficiency alone or combined with iron deficiency resulted in reduced copper concentrations of liver and spleen and reduced ceruloplasmin. Serum triglycerides and cholesterol did not differ among experimental treatments. No significant effects of the interaction between dietary iron and copper on serum lipid levels were found.     

Copper metabolism in iron-deficient maternal and neonatal rats

Groups of rats were fed diets providing 8 ppm iron (-Fe) and 250 ppm iron (+Fe) throughout pregnancy and lactation. In spite of the increase in apparent absorption of iron in pregnant -Fe dams, iron deficiency anemia developed, resulting in decreased iron levels in placenta, amniotic fluid and fetal liver. Copper concentration of amniotic fluid was elevated in -Fe dams. On day 17 of lactation, -Fe dams and their suckling pups had hematologic evidence of iron deficiency. While liver and spleen iron decreased in 17-day-old pups, levels of copper increased. Subcellularly, the greatest increase in hepatic copper in -Fe pups was found in the cytosol, thus the increased copper deposition is not similar to copper loading. Serum ceruloplasmin activity was significantly elevated in -Fe lactating dams and was slightly, but not significantly, increased in -Fe pregnant dams and suckling pups.     

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.     

Effects of developmental iron deficiency and post-weaning iron repletion on the levels of iron transporter proteins in rats

BACKGROUND/OBJECTIVESIron deficiency in early life is associated with developmental problems, which may persist until later in life. The question of whether iron repletion after developmental iron deficiency could restore iron homeostasis is not well characterized. In the present study, we investigated the changes of iron transporters after iron depletion during the gestational-neonatal period and iron repletion during the post-weaning period.MATERIALS/METHODSPregnant rats were provided iron-deficient (< 6 ppm Fe) or control (36 ppm Fe) diets from gestational day 2. At weaning, pups from iron-deficient dams were fed either iron-deficient (ID group) or control (IDR group) diets for 4 week. Pups from control dams were continued to be fed with the control diet throughout the study period (CON).RESULTSCompared to the CON, ID rats had significantly lower hemoglobin and hematocrits in the blood and significantly lower tissue iron in the liver and spleen. Hepatic hepcidin and BMP6 mRNA levels were also strongly down-regulated in the ID group. Developmental iron deficiency significantly increased iron transporters divalent metal transporter 1 (DMT1) and ferroportin (FPN) in the duodenum, but decreased DMT1 in the liver. Dietary iron repletion restored the levels of hemoglobin and hematocrit to a normal range, but the tissue iron levels and hepatic hepcidin mRNA levels were significantly lower than those in the CON group. Both FPN and DMT1 protein levels in the liver and in the duodenum were not different between the IDR and the CON. By contrast, DMT1 in the spleen was significantly lower in the IDR, compared to the CON. The splenic FPN was also decreased in the IDR more than in the CON, although the difference did not reach statistical significance.CONCLUSIONSOur findings demonstrate that iron transporter proteins in the duodenum, liver and spleen are differentially regulated during developmental iron deficiency. Also, post-weaning iron repletion efficiently restores iron transporters in the duodenum and the liver but not in the spleen, which suggests that early-life iron deficiency may cause long term abnormalities in iron recycling from the spleen.     

Perinatal iron deficiency alters the neurochemical profile of the developing rat hippocampus

Cognitive deficits in human infants at risk for gestationally acquired perinatal iron deficiency suggest involvement of the developing hippocampus. To understand the plausible biological explanations for hippocampal injury in perinatal iron deficiency, a neurochemical profile of 16 metabolites in the iron-deficient rat hippocampus was evaluated longitudinally by 1H NMR spectroscopy at 9.4 T. Metabolites were quantified from an 11-24 microL volume centered in the hippocampus in 18 iron-deficient and 16 iron-sufficient rats on postnatal day (PD) 7, PD10, PD14, PD21 and PD28. Perinatal iron deficiency was induced by feeding the pregnant dam an iron-deficient diet from gestational d 3 to PD7. The brain iron concentration of the iron-deficient group was 60% lower on PD7 and 19% lower on PD28 (P < 0.001 each). The concentration of 12 of the 16 measured metabolites changed over time between PD7 and PD28 in both groups (P < 0.001 each). Compared with the iron-sufficient group, phosphocreatine, glutamate, N-acetylaspartate, aspartate, gamma-aminobutyric acid, phosphorylethanolamine and taurine concentrations, and the phosphocreatine/creatine ratio were elevated in the iron-deficient group (P < 0.02 each). These neurochemical alterations suggest persistent changes in resting energy status, neurotransmission and myelination in perinatal iron deficiency. An altered neurochemical profile of the developing hippocampus may underlie some of the cognitive deficits observed in human infants with perinatal iron deficiency.     

Effect of iron therapy on phagocytosis and bactericidal activity in neutrophils of iron-deficient infants

Phagocytosis and bactericidal capacity of neutrophils were measured in 10 iron-deficient infants age 6-23 mo. All infants had hemoglobins less than 11 mg/dL with low saturation of transferrin and serum ferritin but were otherwise in good health. Neutrophil function and iron status were assessed at 0, 3-5, 15, 30, and 90 days of oral iron therapy. Phagocytosis was unaffected in iron deficiency and remained unchanged during therapy. Bactericidal capacity was severely impaired prior to treatment. After 3-5 days of ferrous sulfate administration, there was no significant improvement. At day 15 it returned to normal ranges and remained so at days 30 and 90. The sequence of events suggests that iron does not have a direct effect upon circulating neutrophils but, rather, that it is required during the development of neutrophils in the bone marrow.     

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 X 10(5) 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 iron-deficient 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.     

Mastitis in rural Gambian mothers and the protection of the breast by milk antimicrobial factors

Mastitis was found to be a sizeable clinical problem in a group of lactating Gambian mothers. The mean monthly incidence was 2.6% and repeated episodes of mastitis were common. The role of milk antimicrobial factors in the local defence of the breast against mastitis was investigated by analysis of IgA, IgG, IgM, C3, C4, lactoferrin and lysozyme in the breast milk of 10 mastitis patients. Acute inflammation of the breast was accompanied by the rapid appearance of high concentrations of serum-derived immunoproteins in mastitic milk. Changes in the milk levels of lactose, sodium and transferrin indicated that this was due to a temporary opening of the paracellular pathway. Concentrations of secretory immunoproteins (IgA, lactoferrin and lysozyme) exhibited a delayed response, being elevated one week after the attack of mastitis. The normal milk of mastitis sufferers was significantly deficient in IgA, C3 and lactoferrin when compared with other lactating women suggesting that the former were predisposed to mastitis.     

Neonatal iron deficiency results in irreversible changes in dopamine function in rats

Iron deficiency in human infants and in young animal models produces changes in neural functioning that may be related to monoamine metabolism. This study employed both behavioral and biochemical approaches in a design using cross-fostering to examine alterations in dopamine (DA) function when iron deficiency occurs during the neonatal period. We measured brain Fe, dopamine transporters (DAT) and dopamine receptor density in rats made iron deficient, or not, from postnatal day (PND) 4 to PND 14 or 21. Some pups were then weaned to an iron-deficient diet and others to the control diet to examine the reversibility of these effects. Behaviors related to dopamine function were measured. Dopamine D(2) receptor (D(2)R), D(1)R and iron concentrations were approximately 70, 80 and 30% of control values, respectively, in the nucleus accumbens and striatum in iron-deficient rats at PND 14. The DAT density was also reduced to 50% of control density in the nucleus accumbens but was unchanged in the striatum. By PND 21, there was also a significant 50% lowering of DAT, D(1)R and D(2)R densities in the prefrontal cortex (PFC). Iron repletion at PND 21-49 normalized D(1)R, D(2)R, and DAT levels in the nucleus accumbens, PFC and ventral midbrain but not in the striatum. In summary, neonatal iron deficiency is associated with changes in DA biology that vary with duration of iron deficiency, and are not completely normalized despite replenishment of iron status. Changes in DA-related behaviors that were persistent after postweaning iron repletion suggest the existence of a critical neonatal developmental period that is expressed by alterations in DA functioning.     

Effect of hexachlorobenzene on reproduction in the rat

This study was designed to determine the effect of hexachlorobenzene (HCB) on rat reproduction. Dietary concentrations of 0, 10, 20, 40, 80, 160, 320, and 640 ppm of HCB were fed to Sprague-Dawley rats in a four generation reproduction study. Pregnancy (number of dams whelping/number of dams mated), viability (pups surviving 5 days/pups born alive), and lactation (pups weaned/live pups on day 5 less culls) indices were measured. Body weight gain of pups was monitored. HCB residues were measured in the 21-day-old pups of the F₁a and F₃a generations. The two highest dietary concentrations were toxic to the F₀ generation females and 50% and 20% respectively, died. Sucking pups were particularly sensitive and many died prior to being weaned. The viability index was zero in the F₁a and F₁b generations for pups from dams fed 320 and 640 ppm of HCB and only 55% for the 160 ppm group. The lactation index decreased from 30% for the F₁a and F₁b generation pups to 0 for the F₂a and F₂b generation pups in the 160 ppm group. Also, the lactation index decreased from 93% in the F₁a generation to 40% in the F₃b generation for the 80 ppm group. Birth, 5-day and 21-day body weights of pups were decreased by treatment. HCB residues accumulated in the pups in a dose-related manner. The relative liver weight and aniline hydroxylase activities were increased in weanlings from dams fed 40 ppm of HCB. No gross abnormalities were observed in the pups.     

Effect of feeding cassava root meal on reproduction and growth of rabbits

In an experiment designed to determine the effect of feeding cassava-based diets on reproduction of young rabbit does, 16 New Zealand White does, about 36 weeks old, were fed four diets containing 0, 15, 30 or 45% cassava root meal (CRM) daily plus 200 g of green herbage every other day. After being fed those diets for 42 days, the does were bred to bucks fed standard commercial diet. The does were bred two more times after the pups were weaned at 5 weeks of age. Offspring of the first breeding study were used in an 8-week growth trial by feeding them the same cassava-based diets as their dams. The number of pups per litter at birth, 14, 28 and 35 days postpartum and the live weight of pups at the same ages did not differ significantly between treatments. Similarly, the thiocyanate content of the urine and blood serum of the dams was similar for all treatments. Results of the growth study using the offspring of the first breeding showed the average rate of gain, feed consumption, efficiency of feed utilization, the fresh weight of skin, kidney, liver, heart, pancreas and visceral fat were not significantly different. Also the urine and serum thiocyanate concentrations were not different.     

Type of dietary carbohydrate affects thyroid hormone deiodination in iron-deficient rats.

The interactive effect of iron deficiency and dietary carbohydrate type on growth and thyroid hormone status of Sprague-Dawley rats was studied. Rats were fed either an iron-adequate (approximately 35 micrograms Fe/g) or an iron-deficient (less than 3 micrograms Fe/g) diet that contained 70% carbohydrate. The carbohydrate sources were 100% cornstarch (STARCH), 85.7% cornstarch and 14.3% sucrose (STARCH/SUCR), 71.4% cornstarch, 14.3% sucrose and 14.3% dextrin (DEXTRIN), or 100% sucrose (SUCROSE). After 4 wk, iron-deficient rats weighed less than the iron-adequate rats and were severely anemic. Total food intake was lower in iron-deficient than in iron-adequate animals; it was also significantly lower in SUCROSE-fed animals relative to other carbohydrate groups. Plasma glucose concentrations were significantly higher in iron-deficient rats than in iron-adequate rats, but plasma thyroid hormones, thyroxine and triiodothyronine, and liver thyroxine monodeiodinase activity were lower. Deiodination of reverse triiodothyronine in liver was unaffected by iron deficiency regardless of carbohydrate treatment. The STARCH-fed animals had higher rates of hepatic thyroxine monodeiodinase activity than rats fed the other dietary carbohydrates. The two main conclusions from this study are that thyroid hormone metabolism is altered by iron deficiency regardless of food intake and that the best purified rodent diet for this type of study would contain a mixture of carbohydrate types to avoid the stimulation of thyroxine monodeiodinase by a 70% cornstarch diet.