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Department of Biochemistry and Molecular Biology, University of Minnesota Medical School, Duluth, MN 55812
* To whom correspondence should be addressed. E-mail: jprohask{at}d.umn.edu.
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ABSTRACT |
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 TOP ABSTRACT IntroductionMaterials and MethodsResultsDiscussionLITERATURE CITED |
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Introduction |
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TOPABSTRACTIntroductionMaterials and MethodsResultsDiscussionLITERATURE CITED |
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Recent experiments demonstrated that Cu-deficient rat pups develop lower plasma Fe as a result of multiple mechanisms (5). Impaired ferroxidase function limits placental Fe transfer from dams to pups (6). Cu-deficient dams produce milk with a lower Fe content, continuing the limitation of Fe to the pups (7). Pups develop an impairment in enterocyte Fe transfer due to decreased hephaestin function or expression that further lowers plasma Fe (5). In these anemic Cu-deficient rat pups, plasma ceruloplasmin (Cp) activity is lower at birth; however, liver Fe accumulation does not occur. Interestingly, Cp null mice do not always develop anemia or have lower plasma Fe (8).
In postweaning rats, dietary Cu deficiency lowers Fe absorption in intestinal enterocytes (9). These Cu-deficient rats have lower plasma Fe levels and are anemic. Cp activity in Cu-deficient animals is reduced, possibly impairing the ability to efflux Fe from macrophage and liver stores and thus may contribute to lower plasma Fe (10). Interestingly, postweaning Cu-deficient rats, despite lower plasma Fe, do not have lower brain Fe (3).
In swine, rats, and mice, Fe supplementation has been used as a method to reverse anemia in Cu-deficient animals with mixed results. In swine, additional Fe administered intraperitoneally or i.v. did not affect anemia or plasma Fe levels during postnatal development (11). In rats, Williams et al. (12) found that supplemental Fe increased hemoglobin levels in the Cu-deficient rats but not to control levels. A study by Reeves and DeMars (13) in Cu-deficient postweaning rats demonstrated that Fe supplemented by either diet or injection failed to correct anemia or raise plasma Fe. Injection of Fe in older Cu-deficient mice also did not raise hemoglobin levels compared with Cu injection (14). However, a single Fe injection to suckling Cu-deficient mice did reverse anemia, suggesting that these nursing mouse pups, like rat pups, were Fe deficient (15).
Current experiments were designed to test the hypothesis that lower brain Fe in Cu-deficient rats is due to lower plasma Fe. Following perinatal Cu deficiency, rat pups were treated with additional Fe in the form of Fe dextran injections in an attempt to elevate plasma Fe and brain Fe. A secondary goal was to determine whether hemoglobin concentrations in Cu-deficient pups responded to Fe injections to assess whether anemia can be eliminated or reduced in Cu-deficient rat pups as demonstrated earlier in Cu-deficient suckling mice. Phenotype of the Cu-deficient rat pups following Fe injection was also compared to determine whether abnormal behavior was due in part to Fe deficiency.
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Materials and Methods |
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TOPABSTRACTIntroductionMaterials and MethodsResultsDiscussionLITERATURE CITED |
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Two perinatal nutritional experiments were conducted. Dams were fed a commercial nonpurified Cu- and Fe-adequate diet prior to start of dietary treatment at embryonic d 7 similar to established recent protocols (3). In Expt. 1, 4 dams were Cu+ and 4 dams were Cu–. In Expt. 2, 6 dams were Cu+ and 6 dams were Cu–. In both experiments, litter sizes were culled to 10 pups on postnatal d 0 (P0). In Expt. 2, remaining P20 rat pups were weaned and placed on the same dietary treatment as their dams. All rats had free access to diet and drinking water and were maintained at 24°C with 55% relative humidity on a 12-h-light cycle (0700–1900 light). All protocols were approved by the University of Minnesota Animal Care Committee.
Fe injections. Fe dextran (Sigma) was mixed with 0.22 µm filtered 0.15 mol/L NaCl (saline) to a concentration of 50 mg Fe/L. In Expt. 1, pups were injected at P11 with 20 mg Fe/kg body weight (BW) i.m. in the biceps femoris. The dose was formulated based on a Fe supplementation study in the Belgrade rat (17). In Expt. 2, pups were injected with a higher dose of Fe (45 mg Fe/kg BW) i.m. in the biceps femoris. This was done because of hemoglobin results of Expt. 1 and to pattern total dose based on a previous trial with Cu-deficient rats (13). Injections began at P5 and continued every 4 to 5 d and ended at P18 after 4 doses were given. Cumulative Fe dose ranged between 5 and 7 mg. In both experiments, injection control rats received saline (S) injections. Thus, 4 groups were studied: Cu– Fe injected (Cu–Fe), Cu– S injected (Cu–S), Cu+ Fe injected (Cu+Fe), and Cu+ S injected (Cu+S).
Tissue collection. Male offspring, an Fe injected and S control injected from each litter, were sampled in both experiments. In Expt. 1, tissues were collected at P0 (noninjected pups) and P13. In Expt. 2, tissues were collected at P0, P20, and P26. In Expt. 1, pups were killed by decapitation at P13. Trunk blood was collected in a heparinized tube. Livers and brains were removed for metal analysis. In Expt. 2, pups at P0 were killed by decapitation. At P20, rats were weighed and anesthetized by injection of ketamine/xylazine and perfused with heparin (4 kU/L) PBS, pH 7.4, for 2 min. At P26, rats were weighed then anesthetized with ketamine/xylazine and killed by decapitation. Brain, upper small intestine (15 cm), livers, and blood tissues were harvested from rat pups. Trunk blood was collected in a heparinized tube at P0, P13, and P26. Blood was collected via cardiac puncture at P20. Intestinal lumens were flushed with S to remove digesta, blotted with tissue paper, and dried to constant weight prior to metal analyses. Brains were removed and dissected on a chilled glass plate (18). All tissues were weighed and either processed for biochemical analysis or frozen in liquid nitrogen and stored at –75°C until used.
Biochemical analyses. Hemoglobin was determined spectrophotometrically as metcyanhemoglobin. Activity of the plasma cuproprotein Cp (EC 1.16.3.1) was measured by following oxidation of o-dianisidine at 37°C (19). Tissues and diet were wet-digested with HNO3 (Trace Metal Grade, Fischer Scientific) and residue was dissolved in 0.1 mol/L HNO3. Samples were analyzed for metals by flame atomic absorption spectroscopy (model 1100 B, Perkin-Elmer) (19). Brain Fe data were corrected for blood Fe contamination unless the rats were perfused (3). Plasma Fe was measured by flame atomic absorption spectroscopy (AAS) following treatment with trichloroacetic acid (20). Briefly, plasma was treated with 1 volume of 200 g/L trichloroacetic acid and heated to precipitate contaminating hemoglobin and release transferrin-bound Fe. More than 95% of hemoglobin bound Fe is removed by this technique. Following centrifugation, the supernatant was diluted with 5 volumes of water and then analyzed by AAS.
Cytochrome c oxidase (CCO) activity was measured using a method described previously (19). The CCO activity was measured by monitoring the loss of ferrocytochrome c (initial concentration, 50 µmol/L) at 550 nm. Total protein was estimated using a modified Lowry procedure (21).
Western blotting. Cerebella from P13 rats were diluted with 9 volumes of 0.05 mol/L potassium phosphate buffer (pH 7.0) with protease inhibitors (Protease Inhibitor Cocktail, Sigma) and homogenized. One-half was used for the CCO assay. The sample half for immunoblotting was rehomogenized with 1% Triton X-100 (5 g/L) and then centrifuged at 14,000 x g; 15 min at 4°C. The supernatant was saved for analysis. Western blots were processed in a manner described previously (22). Briefly, 20 µg of protein was loaded on 10% SDS-PAGE gels. After size fractionation, the proteins were transferred to 0.2-µm nitrocellulose membranes. The membranes were incubated with 1:500 dilutions of anti-human TfR (Zymed Labs), anti-human Cu chaperone for superoxide dismutase (CCS) (23), or anti-rabbit lactate dehydrogenase (AB1222, Chemicon International). For CCO subunit IV (COX IV) (AB21348, Molecular Probes), antibody was 1:4000. Apropos secondary antibodies were applied at 1:10,000. Following blot development and capture of the chemiluminescence on film, we performed densitometry (Kodak Image Station 2000MM) and analyzed the band intensities.
Behavior analyses. Vibrissae-elicited unilateral forelimb placement was tested at P25 to compare with recent studies following perinatal Fe deficiency (24). The rat was held by the torso allowing 1 forelimb to hang free. The vibrissae on the ipsalateral side were brushed against a table edge. A successful response occurred when the rat immediately placed the ipsalateral forelimb on the table top. Ten comfortable trials were conducted for each rat on each side. A successful trial was scored as 1. Means were computed after scores were tallied following 20 trials per rat.
Statistics.
Means and SEM were calculated for all data. Variance equality was tested by Bartlett's test, 
= 0.05. Significant unequal variances were detected. Therefore, treatment effects in the 4 groups (diet, injection, or experiment number for P0 pups) were evaluated by 1-way ANOVA and Scheffé F-test, = 0.05. ANOVA data were analyzed using KaleidaGraph (Synergy Software).
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Results |
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Enzyme activity and protein expression were analyzed in the cerebellum of P13 pups (Fig. 2). CCO activity was not affected by Fe injection, with both Cu– groups exhibiting lower CCO activity. Importantly, CCO activity of the Cu–Fe group remained low despite the repletion of brain Fe. The expression of TfR1, used as a marker of Fe deficiency, was significantly affected by Fe injection, as the Cu–S group had higher cerebellar TfR1 levels than all other groups. Two other proteins, known to change in Cu deficiency, were also examined. CCS was affected by dietary treatment only, with both Cu– groups having higher levels of CCS protein. COX IV was also affected only by the dietary treatment. Both Cu– groups had lower levels of COX IV protein (Fig. 2).
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In Expt. 2, a higher dose of Fe dextran was injected, but in neither Expt. 1 or 2 were any adverse reactions to Fe injection detected. Six characteristics were measured in the pups at P20 (Table 3). BW were modestly affected, with the Cu–Fe group means significantly lower than other groups. Cp activity was affected by dietary and Fe injection treatments. Both Cu– groups had lower concentrations than the Cu+ groups. Interestingly, Fe injection lowered Cp activity in the Cu+Fe group. Liver Cu concentrations were also affected by diet and Fe treatment and showed a response similar to Cp activity changes, with both Cu– groups having lower mean concentrations than Cu+ groups and the Cu+Fe group with lower liver Cu concentrations than the Cu+S group. Liver Fe concentrations had a Fe injection treatment effect. Fe-injected groups had much higher concentrations than the S-injected groups. Small intestine Cu concentrations were lower in both Cu– groups. The small intestine Fe concentration showed that the Cu+Fe group had higher Fe concentrations than the Cu+/S group.
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The final tissue collection occurred at P26, 6 d after weaning and 8 d after rats' last dose of injected Fe. Two blood parameters were measured to assess Fe status. Plasma Fe was significantly affected by diet (Fig. 5). Cu– groups had lower plasma Fe levels than the Cu+ groups. Hemoglobin concentrations were higher in Cu+ groups and were affected by Fe injection only in the Cu–Fe group. Brain Fe concentration was affected by diet and Fe injection. The Cu–S group had a lower Fe concentration than the other groups. The Cu–Fe-injected group had brain Fe levels equivalent to Cu+ groups. Brain Cu concentrations were affected by diet treatment only. The Cu– groups had markedly lower Cu concentrations than the Cu+ groups.
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Rats, 10 per treatment group, were tested on vibrissae-elicited foot placement at P25 1 d before sampling (Fig. 6). After a total of 20 trials, Fe injection did not, whereas dietary treatment did, affect foot placement. Both Cu– groups had fewer foot placements compared with the Cu+ groups.
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Discussion |
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TOPABSTRACTIntroductionMaterials and MethodsResultsDiscussionLITERATURE CITED |
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Injection of Fe into Cu-deficient pups raised plasma Fe and in turn brain Fe, demonstrating that pups can utilize extra Fe they acquire. Also, Cu–S pups had higher TfR1 expression in brain than all groups, consistent with Fe deficiency (3). Cu–Fe pups had TfR 1 expression levels equivalent to both Cu+ groups, suggesting that the brain Fe deficiency was eliminated by Fe injection treatment. Interestingly, the restoration of the brain Fe concentration to Cu– pups did not augment the activity of CCO or expression of COX IV. Thus, the reduction in CCO in Cu deficiency is unlikely due to a secondary effect of Fe deficiency. Longer repletion times will be necessary to fully explore this observation. However, data at P26 also did not show an effect of Fe injection. These Cu–Fe pups likely had normal brain Fe from P8 to P26, yet still had 80% lower CCO activity at P26, an effect similar to Cu–S pups. Others have postulated that CCO deficiency is a downstream target of dietary Fe deficiency associated with abnormal behavior (25). Fe injection did not alter the Cu– cerebellar phenotype, as CCS levels remained higher and COX IV subunit levels remained lower compared with Cu+ groups, as documented previously in Cu– rat cerebella (26).
Expt. 2 was designed to deliver a greater load of Fe to perinatal Cu-deficient pups in part because of the hemoglobin response following the single Fe injection in Expt. 1. Following perfusion, to rid the brain of contaminating Fe from blood, brain Fe in Cu– rats injected with Fe was raised in 4 different regions, suggesting that Cu deficiency lowers brain Fe in multiple regions and this defect is corrected by raising plasma Fe. This confirmatory experiment supports recent work that suggests the reason for low brain Fe in Cu– rats is low plasma Fe (4).
Rats display neurological consequences of perinatal Cu deficiency even after long-term Cu repletion (27,28). It was not known if the persistent behavioral abnormalities were caused by perinatal Cu or Fe deficiency. Because Fe concentrations in Cu– rats were raised to levels equal to Cu+ pups, a behavioral test was carried out to determine whether Cu deficiency or Fe deficiency was responsible for changes in neurological function. Previous tests of rats in a perinatal Fe-deficient model showed a decreased response in vibrissae-elicited foot placements, demonstrating an alteration of striatal function (24). In the Cu–Fe rats in the current studies, restoration of brain Fe did not reverse deficit in foot placement behavior. This unique finding suggests Cu deficiency alters hippocampal/striatal behavior and that limiting Fe does not explain the phenotype. Additional studies will be necessary to fully exclude Fe as a confounding variable in the altered behavior created by perinatal Cu deficiency. Prior studies have shown that Cu-deficient rat pups have lesions in the striatum, including lower dopamine concentration (29–31). Altered dopaminergic function has been implicated in the aberrant behavioral phenotype of Fe deficiency (32).
A classic hallmark of Cu deficiency is anemia, which in these studies was defined as lower than normal hemoglobin concentration (33). Hemoglobin concentrations of Cu– rats at all sampling points after the injection of Fe were elevated in both experiments. This was the case in Expt. 2 even though the final sampling point was 8 d postinjection and plasma Fe had dropped. This is consistent with our recent data that suggest the Cu-deficient rat pups are Fe deficient (5). This also confirms our earlier work in Cu– mouse pups injected with Fe (15). However, these results contrast with previous work published by Reeves and DeMars (13) in postweaning rats where no effect of additional Fe administered via diet or injections was seen reversing anemia despite elevating plasma Fe. The Fe dose in those injections studies was similar to those in the current Expt. 2 (5.6 mg total Fe). Work done by Williams et al. (12) with postweaning Cu-deficient rats demonstrated increased hemoglobin levels upon Fe injection. However, this study used an unsupplemented condensed milk diet that was deficient in both Cu and Fe. Cu-replete rats injected with 5 mg Fe had significantly higher hemoglobin levels than Cu-deficient Fe-injected rats. Similarly, Cu-deficient pigs injected with Fe also had no hemoglobin or erythropoietic response (11). Thus, Cu- suckling mammals respond as though Fe deficient, whereas older Cu-deficient mammals cannot reverse anemia even after plasma Fe is restored to control levels, suggesting different age-dependent mechanisms for anemia.
Interestingly, young mice that are Cu deficient have normal plasma Fe and, thus, normal brain Fe concentrations (4). However, these Cu-deficient mice have markedly lower hemoglobin. The anemia observed in Cu-deficient mice may be driven by a different mechanism than the rat even though additional Fe will raise hemoglobin levels in young Cu-deficient mice but not in older Cu-deficient mice (14). Anemia in Cu-deficient mice was purported to be due to impaired hephaestin function and Fe retention in the intestine (34). However, recent work has demonstrated that despite a modest increase in intestinal Fe in Cu-deficient mice, whole-body Fe was not affected by Cu deficiency (4). The mechanism of Cu deficiency anemia in mammals is still unknown and is an area for future work.
An interesting finding in this experiment is that additional injected Fe in the control (Cu+) pups raised hemoglobin above S-injected controls in Expt. 2. Whereas it might appear as though these pups were Fe deficient, it is more likely to be a result of overcoming a physiological block, limiting the amount of Fe transport to the pup. Kochanowski and Sherman (35) showed that hemoglobin levels of rat pups at P20 did not respond to increased dietary Fe to dams as high as 150 mg/kg, likely because milk Fe levels did not respond to dietary Fe. When diet enteral Fe supplements are increased through gastromy to suckling Fe-adequate pups, hemoglobin increases (36). Thus, suckling pups, even though Fe adequate, can respond with higher hemoglobin if the milk-gut barrier is challenged.
Current injection data and previous rodent data support the hypothesis that low brain Fe is the result of low plasma Fe (4). These experiments were designed to create a severe Cu deficiency that resulted in anemia. However, anemia is not always apparent in Cu-deficient rats, even when plasma Fe is low, as in Cu-deficient rat dams (5). The real risk during neonatal development is low plasma Fe, because it potentially limits Fe transport to brain. It is well established that neonatal Fe deficiency is a risk for permanent cognitive impairment in humans (37). Abnormal behavior can exist without anemia in infancy. It is assumed that Cu-deficient human infants with low plasma Fe might be at risk for brain Fe deficiency. Initial identification of Cu-deficient infants reported low plasma Fe in addition to low plasma Cu (38). These facts emphasize the need for accurate trace metal nutritional assessment during infancy and prompt treatment should Cu or Fe deficiency be detected.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Author disclosures: J. W. Pyatskowit and J. R. Prohaska, no conflicts of interest.
3 Abbreviations used: AAS, atomic absorption spectroscopy; BW, body weight; CCS, Cu chaperone for superoxide dismutase; Cp, ceruloplasmin; CCO, cytochrome c oxidase; COX IV, cytochrome oxidase subunit IV; Cu+S, copper-adequate saline injected; Cu-S, copper-deficient saline injected; Cu+Fe, copper-adequate iron injected; Cu-Fe, copper-deficient iron injected; P, postnatal day; S, saline; TfR1, transferrin receptor 1.
Manuscript received 16 May 2008. Initial review completed 12 June 2008. Revision accepted 29 July 2008.
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