Effect of Early Iron Deficiency in Rat on the γ-Aminobutyric Acid Shunt in Brain

Early iron deficiency in rat does not affect the weight or the protein, DNA, and RNA content but results in a slight reduction in gamma-aminobutyric acid (GABA) (13%, p less than 0.01) and glutamic acid (20%, p less than 0.001) content of the brain. The activities of the two GABA shunt enzymes, glutamate dehydrogenase and GABA-transaminase, and of the NAD+-linked isocitrate dehydrogenase (ICDH) were inhibited whereas the glutamic acid decarboxylase, mitochondrial NADP+-linked ICDH, and succinic dehydrogenase activities remained unaltered in brain. On rehabilitation with the iron-supplemented diet for 1 week, these decreased enzyme activities in brain attained the corresponding control values. However, the hepatic nonheme iron content increased to about 80% of the control, after rehabilitation for 2 weeks. A prolonged iron deficiency resulting in decreased levels of glutamate and GABA may lead to endocrinological, neurological, and behavioral alterations.     

Carnitine depletion in rat pups from mothers given mildronate: a model of carnitine deficiency in late fetal and neonatal life

Mildronate (3-(2,2,2,-trimethylhydrazinium)propionate), is a butyrobetaine analogue that is known to inhibit gamma-butyrobetaine hydroxylase, the enzyme catalyzing the last step of carnitine biosynthesis. When administered to adult rats it determines a systemic carnitine deficiency and may therefore serve as an animal model for human carnitine depletion. The aim of this study was to evaluate the effect of mildronate administration to pregnant and lactating rats on tissue carnitine concentrations in 4- and 13-day-old rat pups. At 14 days of gestation female rats began to receive mildronate in the diet (200 mg/kg/d) and this continued for entire lactation period. Mildronate treatment determined a large reduction of carnitine levels in the milk of lactating dams. Because organ carnitine concentrations in neonatal rats are directly related to dietary supply, pups from mildronate group had significantly depleted levels of total carnitine in serum, heart, liver, muscle, brain and pancreas relative to controls, at 4 and 13 days of age. Correspondingly, an increase in triglyceride levels was observed in liver, heart and muscle of mildronate pups. This is in agreement with a reduction of basal rate of oxidation of [U-(14)C]-palmitate to (14)CO(2) and (14)C-acid-soluble products observed in liver homogenates from carnitine-deficient pups. All functional and biochemical modifications were compatible with a carnitine deficiency status. In conclusion our results describe a model of carnitine depletion in pups, suitable for the investigation of carnitine deficiency in fetal-neonatal nutrition, without any concomitant mildronate-mediated metabolic alterations.     

Effects of maternal thiamine deficiency on the lipid composition of rat whole brain, gray matter and white matter

Studies were made of the effects of maternal thiamine deficiency on rat whole brain, gray matter and white matter lipids. Mothers were fed a high protein diet (controls) or thiamine deficient high protein diet (thiamine deficient, TD) from 14th day of gestation through lactation. An additional group (pair fed control, PFC) was pair fed with the thiamine deficient group. The TD pups started showing symptoms of abnormalities in posture, arched back and hind limb paralysis from 16th day of lactation. Significant deficits were found in body weight and brain weight of TD and PFC pups. But the deficits seem to be more in the former group. Significant deficits were observed with regard to the concentration of lipids such as galactolipids, phospholipids and plasmalogens in the whole brain of TD and PFC pups at 21 days of age. Additional deficits were also found in the concentration of cholesterol in PFC pups. Gray matter lipids from TD pups seem to be completely spared. However, deficits were found in galactolipid and ganglioside concentrations in PFC pups. The deficits found in the concentration of different lipids in white matter are similar to those observed in whole brain. These results suggest that the effects of thiamine deficiency may be partly due to resultant growth retardation and partly due to the deficiency of thiamine per se.     

Dietary iron and the integrity of the developing rat brain: a study with the artificially-reared rat pup.

Inadequate iron nutrition is thought to affect many aspects of brain development. Iron is a component of enzyme systems in DNA synthesis, the respiratory chain, neurotransmitter and lipid metabolism. The iron content of the striatum increases post-natally, with neuronal differentiation, myelin lipid and receptor formation: Seventy percent of the iron in the brain is associated with myelin. In an attempt to dissociate the global effects of under-and/or malnutrition and to produce exclusively an iron deficiency, we have used the gastrostomy-reared rat pup fed milk substitutes which vary only in their iron content. To ensure the pups did not have adequate iron reserves at birth, dams were fed a meal diet of low iron content (3 ppm) throughout gestation. The pups were then artificially reared on milk with (43 ppm), and without added iron (2.5 ppm) from 6 up to 21 days after birth. At 21 days of age, body weights of iron deficient pups were about 90% those of control animals. At 21 days of age, the pups were weaned, then fed standard laboratory rat chow. Brain was examined at 42 days of age (for young adults) and up to 6 months of age (180 days as mature adults). Morphometric analysis of sagittal sections of the cerebellum at 21 and 63 days of age revealed a deficit in white matter formation in pups fed low-iron at 21 days of age when compared to controls. This deficit was partially recouped by age 63 days. By contrast, animals fed milk supplemented with iron showed greater definition in white matter formation than controls at 21 days of age; indicative of precocious maturation of the white matter tracts. Our findings indicate that iron deficiency, without under/mal-nutrition and other variables, does not result in extensive growth deficits in body and brain weight. However, the iron status profoundly influences the development of myelination in that the process is delayed in iron deficiency.     

Acetylcholinesterase activity in developing rat brain during undernutrition

The effect of low protein diet on rat brain AChE activity has been studied during gestation, lactation and postweaning periods. There was decrease in enzyme activity of pups undernourished either during gestation and lactation or lactation alone, the decrease being maximum in 18-day-old pups. In postweaning rats, a significant decrease was observed after 2 and 4 weeks of undernutrition compared to the control. However, the effect of undernutrition was annuled by 2-week rehabilitation, thereby indicating that imposed undernutrition only delays the normal level of the enzyme. Moreover, it appears that the enzyme activity depends both on the nutritional status and the development age.     

Rat small intestinal morphology with special reference to villi: effects of maternal protein deficiency and hydrocortisone

The heights of villi were less in all the segments of mildly undernourished (by increasing litter size) pups at 21 days of age, in comparison with controls, while the heights of villi in the duodenum and jejunum, but not in the ileum, were less in severely undernourished (by maternal protein deficiency) pups. Administration of hydrocortisone during 18--21 days of age to normal pups and to pups undernourished by maternal protein deficiency during lactation showed a marked reduction of villi heights in the ileum but not in other segments of the intestine. When the pups of protein-deficient mothers (during lactation) were fed on a normal diet during the post-weaning period, the villi in the ileum were found to be longer than those of controls. Superimposition of post-weaning protein deficiency over pre-weaning undernutrition only left the heights of ileal villi unaltered, but in other segments deficits in heights of villi were observed. These studies suggest that the suggested 'villi-reducing factor' did not appear in the ileum of severely undernourished pups even after the availability of an adequate nutrition in the post-weaning period, probably due to hydrocortisone deficiency at about 21 days of age, and that the hormone has a role in maintaining ileal villi heights. In addition to the heights of villi, other morphological retardations and alterations of enterocyte structure were observed in the severely undernourished intestine. These effects differed in different parts of the intestine and with the degree of neonatal undernutrition. Most of the morphological abnormalities showed a trend for recovery when a normal diet was given in the post-weaning period.     

Copper deficient rats and mice both develop anemia but only rats have lower plasma and brain iron levels

Iron homeostasis depends on adequate dietary copper but the mechanisms are unknown. Mice (Mus musculus) and rat (Rattus norvegicus) offspring were compared to determine the effect of dietary copper deficiency (Cu-) on iron status of plasma, liver, brain and intestine. Holtzman rat and Hsd:ICR (CD-1) outbred albino mouse dams were fed a Cu- diet and drank deionized water or Cu supplemented water. Offspring were sampled at time points between postnatal ages 13 and 32. Cu- rat and mouse pups were both anemic, but only rat pups had lower plasma and brain iron levels. Plasma iron was lower throughout the suckling period in Cu- rats but not Cu- mice. Cu- mice derived from dams restricted of Cu only during lactation were also severely anemic without hypoferremia. Intestinal metal analysis confirmed that Cu- pups had major reductions in intestinal concentration of Cu, increased Fe, and normal Zn. However, whole mouse (less the intestine) analysis demonstrated normal content of Fe indicating that the limitation in iron transport by intestinal hephaestin had no consequence to total iron reserves of the mouse. Further research will be needed to determine the reason Cu- mice were anemic since the "ferroxidase" hypothesis does not explain this phenotype.     

Effects of marginal zinc deficiency on microtubule polymerization in the developing rat brain

One of the possible mechanisms that has been proposed to underlie the deleterious effects of zinc deficiency on brain development is an impairment in the normal formation of the cytoskeletal network. In the current study, in vivo microtubule polymerization was characterized in brain supernatant fluids, from 20-d-old pups whose dams were fed diets containing control (50 micrograms zinc/g) or marginal levels of zinc (10 micrograms zinc/g) throughout pregnancy and lactation. Pup brain and body weights were similar between the groups; however, plasma zinc concentrations were lower (27%) in pups fed the marginal zinc diet than in controls. Tubulin concentrations in 100,000 g brain supernates were similar between the groups; however, tubulin polymerization in the brain supernates was significantly lower in pups fed the marginal zinc diet compared to controls. Primarily, the early events of polymerization were affected; the lag period of the reaction was doubled, and the initial velocity was slower (26%) in supernates from pups fed the marginal zinc diet than in controls. These findings support the idea that some of the negative effects of marginal zinc deficiency on brain development and function may be mediated by an alteration in microtubule formation.     

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.     

Dose-effect of dietary oleic acid: oleic acid is conditionally essential for some organs

The minimum dietary intake of oleic acid that is indispensable to maintain a normal content of this fatty acid in several tissues (heart, muscle, kidney and testis) was determined in the rat. For this purpose, a dose-effect study was conducted using an experimental protocol with 7 groups of rats who received a diet in which the oleic acid level varied from 0 to 6000 mg per 100 g diet, but the other ingredients were identical (in particular the essential fatty acids, linoleic and alpha-linolenic acid). Female rats were fed the diets from two weeks before mating, and their pups were killed aged either 21 or 60 days. When the level of oleic acid in the diet was increased, the main modifications observed in 21-day-old deficient pups were as follows: (i) for 18:1n-9, in the liver, muscle, heart, kidney, and testis, a plateau was reached at about 4 g oleic acid per 100 g diet. Below this level, the higher the dose the greater the response; (ii) for 16:1n-7, the concentration decreased in the liver, muscle, heart, kidney and testis; (iii) the concentration of 18:1n-7 decreased in the kidney, muscle, and testis; (iv) some minor modifications were noted for the other fatty acids. In mother's milk at 14 days of lactation, when dietary oleic acid increased, the levels of 18:1(n-9) also increased; the increase was regular and did not reach a plateau. In 60-day-old rats, the results were generally similar to those in 21-day-old rats, but with some differences, in particular a slight decrease in oleic acid concentration in the liver and kidney at the highest dietary oleic acid level.     

Multiple mechanisms account for lower plasma iron in young copper deficient rats

Copper deficiency lowers brain copper and iron during development. The reduced iron content could be due to hypoferremia. Experiments were conducted to evaluate plasma iron and “ferroxidase” hypotheses by determining copper and iron status of Holtzman albino rats following gestational/lactational copper deficiency. Copper deficient (Cu−) dams on treatment for 5 weeks, two of gestation and three of lactation, had markedly lower copper content of milk and mammary tissue, and lower milk iron. Newborn pups from Cu− dams had lower copper and iron concentrations. Compared to Cu+ pups, Cu− pups, analyzed between postnatal age (P) 0 and P26, were smaller, anemic, had lower plasma iron, cardiac hypertrophy, and near zero ceruloplasmin activity. Liver copper in Cu+ pups increased then decreased during development and major reductions were evident in Cu− pups. Liver iron in Cu+ pups decreased with age while nursing but increased after eating solid food. Liver iron was lower in Cu− pups at P0 and P13 and normal at P20 and P26. Small intestinal copper decreased with age in Cu+ pups and was lower in Cu− pups. Intestinal iron levels in Cu− pups were higher than Cu+ pups postweaning in some experiments. Reduction in plasma iron in Cu− pups is likely due to a decreased “ferroxidase” function leading to lower placental iron transport, a lower milk iron diet, and partial block in iron uptake from intestine but is not due to failure to mobilize hepatic iron, in contrast to older rats eating diet with adequate iron.     

Effects of maternal marginal zinc deficiency on myelin protein profiles in the suckling rat and infant rhesus monkey

In the current study, the effects of marginal Zn deficiency on myelin protein profiles in neonatal rats and rhesus monkeys were investigated. Following mating, rats were fed a Zn-adequate diet,ad libitum (50 μg Zn/g; 50 Zn AL), or a marginal Zn diet (10 μg Zn/g) from day 0 (10 Zn d0) or day 14 (10 Zn d14) of gestation to day 20 postnatal. An additional group of dams was restricted-fed the control diet to the food intake of the 10 Zn d0 group (50 Zn RF). Day 20 pup plasma and liver Zn concentrations in the 10 Zn groups were lower than in the 50 Zn groups. In a parallel experiment, rhesus monkeys were fed a Zn-adequatead libitum diet (100 μg Zn/g) or a marginal Zn diet (4 μg Zn/g diet; MZD) throughout gestation and lactation. Day 30 monkey infant plasma and liver Zn levels were similar in the MZD and control groups. Rat brain and monkey brain cortex weights were similar among the dietary groups. The amount of myelin recovered (mg protein/g brain) from day 20 rat pups from the 10 Zn groups was lower than that recovered from the 50 Zn rat pups. Myelin recovery from the MZD and control monkey infants was similar. When myelin protein profiles were characterized, it was found that the percentages of high-molecular-weight (HMW) proteins and Wolfgram protein were higher, whereas the percentages of small and large basic proteins were lower in myelin from the 10 Zn d0 and 50 Zn RF pups compared to the distribution in the 50 Zn AL rat pups. Results for the 10 Zn d0 and 10 Zn d14 pups were similar for all of the parameters studied. The percentage of HMW proteins was higher and that of basic protein lower in myelin from MZD monkey infants compared to the percentage of these proteins in myelin from controls. Although the interpretation of the rat data is complicated because of the anorexia associated with the Zn deficiency, the observed changes in monkey myelin protein profiles provide strong evidence that maternal Zn deficiency affects myelination in the offspring.     

Pre- and postnatal protein undernutrition increases hepatic carnitine palmitoyltransferase I activity and decreases enzyme sensitivity to inhibitors in the suckling rat.

Rats were pair-fed isocaloric diets containing either 25% (control diet) or 6% protein (low-protein diet) during the 5 weeks prior to conception and through the gestation and lactation periods; then, carnitine palmitoyltransferase I (CPT-I) activity was determined in liver and skeletal muscle mitochondria isolated from the corresponding pups. Maternal protein undernutrition increased the activity of hepatic CPT-I all along the suckling period, whereas the activity of the skeletal muscle enzyme was unaffected. Moreover, the sensitivity of hepatic CPT-I to inhibition by both malonyl-CoA and 4-hydroxyphenylglyoxylate was decreased in the low-protein group. These alterations in the properties of hepatic CPT-I may be involved in the appearance of hyperketonemia in the rat pup upon maternal administration of low-protein diets.     

Spatial memory deficits in maternal iron deficiency paradigms are associated with altered glucocorticoid levels

"The goal of this study was to examine the effect of maternal iron deficiency on the developing hippocampus in order to define a developmental window for this effect, and to see whether iron deficiency causes changes in glucocorticoid levels. The study was carried out using pre-natal, post-natal, and pre + post-natal iron deficiency paradigm. Iron deficient pregnant dams and their pups displayed elevated corticosterone which, in turn, differentially affected glucocorticoid receptor (GR) expression in the CA1 and the dentate gyrus. Brain Derived Neurotrophic Factor (BDNF) was reduced in the hippocampi of pups following elevated corticosterone levels. Reduced neurogenesis at P7 was seen in pups born to iron deficient mothers, and these pups had reduced numbers of hippocampal pyramidal and granule cells as adults. Hippocampal subdivision volumes also were altered. The structural and molecular defects in the pups were correlated with radial arm maze performance; reference memory function was especially affected. Pups from dams that were iron deficient throughout pregnancy and lactation displayed the complete spectrum of defects, while pups from dams that were iron deficient only during pregnancy or during lactation displayed subsets of defects. These findings show that maternal iron deficiency is associated with altered levels of corticosterone and GR expression, and with spatial memory deficits in their pups."     

Development of hepatic and adipose tissue lipogenic enzymes and insulinemia during suckling and weaning on to a high-fat diet in Zucker rats

This study was designed to monitor the developmental changes in insulinemia and lipogenic enzyme activities in both inguinal adipose tissue and liver during suckling (7, 9, 14, and 17 days of age) and weaning (22 and 30 days of age) on to either a low-fat or a high-fat diet in lean (Fa/fa) and obese (fa/fa) rats. Tissues were removed through surgery and genotypes were retrospectively determined. During suckling, there was no difference in liver enzyme activities between the two groups. In contrast, adipose tissue fatty acid synthetase was increased by 50% and citrate cleavage enzyme and malic enzyme by 30% by 9 days of age. By 17 days of age, there was a threefold elevation in these enzyme activities and 6-phosphogluconic dehydrogenase and a twofold increase in glucose-6-phosphate dehydrogenase per inguinal fat pad in fa/fa versus Fa/fa. Consistent with these results, fat pad weight was increased by 20%, 50%, and 100% at 9, 14, and 17 days of age, respectively, in obese as compared to lean pups. However only by 17 days of age could a slight but significant increase in insulin level be detected in obese pups. Enlargement of inguinal fat pad accelerated after weaning on to a low-fat diet and still more after weaning on to a high-fat diet. Weaning on to a low-fat diet elicited an induction of hepatic lipogenic enzymes two or three times greater in fa/fa than in lean pups, while weaning on to a high-fat diet blunted the differences between genotypes. The lipogenic enzyme activities displayed per total inguinal fat were three to ten times greater in obese than in lean pups, regardless of the diet. However, adipose tissue lipogenic enzyme activities were much lower after weaning on to a high-fat than on to a low-fat diet in obese pups. The high-fat diet was as effective as the low-fat diet in triggering hyperinsulinemia in obese pups. The increased adipose tissue capacity for lipogenesis, starting during the suckling period, could play an important etiologic role in the development and maintenance of obesity in the Zucker rat.-Bazin, R., and M. Lavau. Development of hepatic and adipose tissue lipogenic enzymes and insulinemia during suckling and weaning on to a high-fat diet in Zucker rats.     

Effects of age and biotin status on postnatal development of plasma biotinidase activity in rats

Biotinidase activity was measured in plasmas of 1-, 7-, 14-, and 21-day-old rats from control dams and dams that had been fed a biotin-depleting diet from Day 15 of gestation. Biotinidase activity increased significantly in the plasma of rats from control and depleted mothers until Postnatal Day 14, after which there was a small but significant decline at Day 21. Differences between the mean activities of the two groups of pups on each sampling day were not significant and there were no significant differences in activity levels attributable to sex. Plasma albumin concentrations increased from birth until Day 21, and plasma biotinidase activity and albumin concentration were significantly correlated (r = +/- 0.43). We suggested that these two proteins may be controlled by a common mechanism in the early postnatal period, and that biotin deficiency does not affect the development of biotinidase activity. Because biotin-depleted neonatal pups show developmental changes in biotinidase activity similar to those of human newborns, and they can be produced reliably by depleting dams from Day 15 of gestation, they may be useful models for studying the developmental abnormalities associated with human biotinidase deficiency.     

Neuroleptic-Induced Supersensitivity and Brain Iron: I. Iron Deficiency and Neuroleptic-Induced Dopamine D2 Receptor Supersensitivity

Previous studies have shown that nutritional iron deficiency in rats reduces brain iron content, resulting in dopamine D2 receptor subsensitivity, as indicated by a decrease in [3H]spiperone binding in caudate nucleus and in behavioral responses to apomorphine. Both phenomena can be reversed by iron supplementation. The possibility that neuroleptic-induced dopamine D2 receptor supersensitivity involves an alteration in brain iron content was investigated in nutritionally iron-deficient and control rats chronically treated with haloperidol (5 mg/kg daily for 14 or 21 days). Neuroleptic treatment was initiated either (a) concurrently with iron deficiency or (b) 2 weeks after the start of iron deficiency. The results show that dopamine D2 receptor subsensitivity, a feature of iron deficiency, is absent in haloperidol-treated, iron-deficient groups. On the contrary, these animals demonstrated biochemical and behavioral dopamine D2 receptor supersensitivity that is relatively greater than that observed with control, haloperidol-treated animals. Haloperidol (5 mg/kg daily for 21 days) as well as chlorpromazine (10 mg/kg daily for 21 days) caused a significant reduction (20-25%) in liver nonheme iron stores as compared with values in control rats. However, in iron-deficient rats, in which liver iron stores were almost totally depleted, haloperidol had no effect. The ability of chronic haloperidol treatment to prevent the reduction of dopamine D2 receptor number during iron deficiency may be associated with alteration of body iron status. Thus, less iron may result in an increase in free haloperidol available to the dopamine D2 receptor.     

Effect of protein deficiency in the female rat diet during pregnancy and lactation on the activity of the membrane and soluble forms of digestive enzymes in the offspring small intestine

Protein deficiency in female rats diet during pregnancy and lactation resulted in deceleration of induction of sucrase both forms in the jejunum and ileum; in acceleration of induction of the maltase membrane from in the jejunum; and in suppression of the lactase membrane form in the ileum; in earlier forming of the adult-type distribution of activity of the membrane form of intestinal alkaline phosphatase and in a decrease in activity of the enzyme soluble form. The findings are corroborated by a suppression of activities of the membrane and soluble forms of the small intestine digestive enzymes in 30-day old rat pups fed with a control (adequate) ration starting 21 days after the birth.     

Effects of neonatal pantothenic acid deficiency on brain lipid composition in rats.

—Studies were made of the effects of pantothenic acid deficiency during the neonatal period on brain lipids in rats. Mothers with 6–8 pups to a litter were fed from soon after birth a diet either normal or deficient in pantothenate. An additional control group (restricted controls) was pair-fed with the deficient group. Significant deficits were found in the pups of the pantothenate-deficient group and in those of the restricted controls with regard to body weight, brain weight and brain concentration of lipids (total lipid, cholesterol, phospholipid, galactolipid and gangliosides) at 21 days of age. The deficits in both these groups were comparable. The results suggest that the effects of pantothenate deficiency may be due to the resulting growth deficit rather than to the deficiency of pantothenate per se.