Signs of iron deficiency in copper-deficient rats are not affected by iron supplements administered by diet or by injection.

The goal of this study was to determine the effects of Fe supplementation on the anemia of Cu deficiency in rats. In addition, we observed changes in serum and organ Cu and Fe during the development of Cu deficiency. In Experiment 1, weanling male Sprague-Dawley rats were fed AIN-93G diets containing either <0.3 mg Cu [Cu deficient (CuD)] or 6.0 mg Cu [Cu adequate (CuA)] per kilogram diet, and 35 mg Fe/kg. Five rats from each group were killed at intervals for the analysis of hematologic parameters and mineral content of various organs. In Experiment 2, two groups of 24 rats each were fed either the CuA diet or the CuD diet for 14 days. Then, three sets of eight rats in each group received three separate Fe treatments: (1) daily intraperitoneal injections of 400 mug Fe (Cu-free ferric citrate) per rat for another 14 days, (2) fed similar diets that contained three times the normal amount of Fe (105 mg/kg) for 14 days, or (3) received no further Fe treatment. At day 21, all rats were fed a 1-g meal labeled with (59)Fe to determine Fe absorption. After 28 days, rats were killed for the analyses of Fe and Cu status. Results of Experiment 1 showed that within 14 days, CuD rats had lower blood hemoglobin (Hgb), red blood cell count, and mean corpuscular volume than CuA rats. Copper concentrations in all tissues measured were lower in the CuD rats than in controls. Serum ceruloplasmin (Cp) activity in CuD rats was only 0.8% of CuA rats at day 7. During this period, enterocyte and liver Fe concentrations were elevated and serum Fe was reduced, but there was no change in spleen Fe. Results of Experiment 2 showed that CuD rats absorbed less Fe than CuA rats. Supplemental Fe by diet or by intraperitoneal injections did not prevent anemia in the CuD rats or affect other parameters of Cu status. Serum total iron binding capacity [transferrin (Tf)] was not changed by Cu deficiency or by Fe supplementation; however, percent Tf saturation was reduced in CuD rats but was not enhanced by Fe supplementation. These data suggest that anemia of Cu deficiency occurs because of reduced Fe absorption, and it inhibits release of Fe from the liver and inefficient loading of Fe into Tf because of very low plasma Cp activity. The latter then leads to inefficient delivery of Fe to the erythroid cells for heme and Hgb synthesis.

Repletion of copper-deficient rats with dietary copper restores duodenal hephaestin protein and iron absorption.

Copper (Cu) deficiency in rats reduces the relative concentration of duodenal hephaestin (Hp), reduces iron (Fe) absorption, and causes anemia. An experiment was conducted to determine whether these effects could be reversed by dietary Cu repletion. Five groups of eight weanling male rats each were used. Group 1 was fed a Cu-adequate diet (5.0 mg Cu/kg; CuA) and Group 2 was fed a Cu-deficient diet (0.25 mg Cu/kg; CuD) for 28 days. The rats were fed 1.0 g each of their respective diets labeled with 59Fe (37 kBq/g), and the amount of label retained was measured one week later by whole-body-counting (WBC). Group 3 was fed a CuA diet and Groups 4 and 5 were fed a CuD diet for 28 days. Group 5 was then fed the CuA diet for another week while Groups 3 and 4 continued on their previous regimens. Rats in Groups 3, 4, and 5 were fed 1.0 g of diet labeled with 59Fe, and the amount of label retained was measured by WBC one week later. Rats were killed and duodenal enterocytes isolated for Hp protein analysis, whole blood was analyzed for hematological parameters, and various organs for 59Fe content. CuD rats absorbed less (P<0.05) Fe than CuA rats, the relative amount of duodenal Hp was less (P<0.05) in CuD rats, and the CuD rats developed anemia. After the CuD rats had been repleted with Cu for one week, Fe retention rose to values even higher (P<0.05) than those in CuA rats. After two weeks, the relative amount of duodenal Hp was higher (P<0.05) than normal, and most signs of anemia were reversed. Liver 59Fe was elevated in CuD rats, but was restored to normal upon Cu repletion. These findings suggest a strong association between duodenal Hp abundance and Fe absorption in the CuD rat, and that reduced Fe absorption is an important factor in the cause of anemia.

Dietary copper deficiency reduces iron absorption and duodenal enterocyte hephaestin protein in male and female rats.

The mechanism for reduced Fe absorption in Cu deficiency is unknown, but may involve the intestinal Cu-dependent ferroxidase, Hephaestin (Hp). A 2 x 2 factorial experiment was designed to include Cu-deficient (CuD) and Cu-adequate (CuA) male and female rats. Weanling rats of both sexes were randomly divided into 2 groups each and fed an AIN-93G diet with low (<0.3 mg/kg; CuD) or adequate Cu (5.0 mg/kg; CuA). After 19 d, rats were fed 1.0 g each of their respective diets labeled with (59)Fe. Retained (59)Fe was monitored by whole-body counting for 12 d. Then, rats were killed for (59)Fe and Fe measurements in blood and various organs. Duodenal enterocytes were isolated for Western blot analysis of Hp. Signs of Cu and Fe deficiency were evident in both sexes. CuD male rats absorbed 60% as much Fe as CuA male rats (P < 0.001), whereas CuD female rats absorbed 70% (P < 0.001) as much as CuA females, with no difference between the sexes. Hp protein in enterocytes of CuD rats of both sexes was only 35% of that in CuA rats. The biological half-life of (59)Fe in CuD rats was only 50% (P < 0.001) of that in CuA rats, suggesting that Fe turnover was faster in CuD rats than CuA rats. Serum, spleen, and kidney Fe were lower (P < 0.001) in CuD rats than in CuA rats. Duodenal mucosa and liver Fe were higher (P < 0.01) in CuD male rats than CuA rats. Duodenal Fe but not liver Fe was higher in CuD female rats than CuA rats. Liver Fe was much higher (<0.001) overall in females than males. The data suggest that Cu deficiency reduces Fe absorption in rats through reduced expression of duodenal Hp protein.

Copper deficiency reduces iron absorption and biological half-life in male rats.

Dietary copper deficiency (CuD) in rats leads to iron (Fe) deficiency anemia. Is this because CuD reduces Fe absorption? Fe absorption in CuD rats was determined by feeding diets labeled with (59)Fe and using whole-body counting (WBC) to assess the amount retained over time. Two groups, each with 45 male weanling rats, were fed an AIN-93G diet low in Cu (<0.3 mg/kg; CuD) or one containing adequate Cu (5.0 mg/kg; CuA). At intervals over the next 42 d, 5 rats per group were killed and blood was drawn to determine hematocrit, hemoglobin, and other indicators of Fe status. At d 7 and 25, 5 rats per group were fed 1.0 g of their respective diets that had been labeled with (59)Fe. Retained (59)Fe was monitored for 10 d by WBC; then rats were killed and (59)Fe was measured in various organs. Signs of Fe deficiency, such as low hemoglobin, hematocrit, and RBC count, were evident in CuD rats by d 14. At d 7, CuD rats absorbed 90% as much Fe as CuA rats (P > 0.20), but at d 25, CuD rats absorbed only 50% as much as CuA rats (P < 0.001). In the study beginning at d 7, the biological half-life (BHL) of (59)Fe in CuD rats was less (P < 0.02) than that in CuA rats [geometric mean (-SEM, +SEM); 75(62,91) d vs. 175(156,195) d]. In the study beginning at d 25, the BHL was again less (P < 0.02) in the CuD rats than in the CuA rats [33(23,49) d for CuD and 157(148,166) d for CuA]. Apparently, the route of Fe loss in the CuD rats was through the gut. At d 16 and 34, CuD rats lost 4 to 5 times more (P < 0.01) (59)Fe in the feces in a 24-h period than the CuA rats. Also, (59)Fe in the duodenal mucosa of CuD rats was approximately 100% higher (P < 0.01) than in CuA rats. These findings suggest that Fe deficiency anemia in CuD male rats is caused at least in part by reductions in Fe absorption and retention.

Increased heme oxygenase-1 expression during copper deficiency in rats results from increased mitochondrial generation of hydrogen peroxide.

The activity of hepatic heme oxygenase (HO) in rats is elevated in response to copper deficiency. However, the mechanism responsible for the increase in HO activity is poorly understood. Oxidative stress is a common denominator for many of the signals that induce HO-1, the inducible isoform of HO. The present study evaluated the role of H(2)O(2) and the mitochondrial electron transport chain as a potential mechanism for the induction of HO-1 during copper deficiency. Mitochondria isolated from the livers of young male rats fed a copper-deficient diet for 5 wk had significantly (P < 0.05) reduced levels of NADH:cytochrome c reductase (31% reduction), succinate:cytrochrome c reductase (42% reduction), and cytochrome c oxidase (70% reduction) activities and significantly increased production of H(2)O(2) (48% increase) when glutamate was used as a substrate. Hepatic levels of HO-1 protein and mRNA were also significantly elevated (48 and 20%, respectively) in copper-deficient rats, indicating that copper deficiency stimulated the expression of the HO-1 gene. Furthermore, hepatic HO-1 protein content was best described by a regression model that included mitochondrial NADH:cytochrome c reductase and succinate:cytochrome c reductase activities, but not cytochrome c oxidase activity (R(2) = 0.54, P < 0.02). Hydrogen peroxide is a known inducer of HO-1, and our results suggest that increased mitochondrial H(2)O(2) production resulting from inhibition of respiratory complex activities contributes to the induction of HO-1 during copper deficiency. The levels of HO-1 protein and mRNA were also elevated (85 and 95%, respectively) in hearts from copper-deficient rats, indicating that the effects of copper deficiency on HO-1 gene expression are not limited to hepatic tissue.