High‐Iron Intake Impairs Growth and Causes Copper‐Deficiency Anemia in Mice

Abstract

A commonly used method to cause iron loading in rodents is feeding of a 1–2% carbonyl iron diet. This high amount of dietary iron presumably bypasses normal protective mechanisms and leads to increased body iron levels. We utilized this dietary iron‐overload model in a mouse feeding study in which we also altered the dietary copper content. This study was intended to test the hypothesis that dietary copper would influence intestinal iron absorption during iron overload. Weanling, male C57BL/6 mice were housed in overhanging, wire mesh‐bottomed cages and fed one of 6 different AIN‐93G‐based diets for 4 weeks containing normal (79 ppm) or high (8820 ppm) iron in combination with low (0.9 ppm), normal (8 ppm) or high (183 ppm) copper. Diets were otherwise identical. All data reported below were from 4–8 mice per group and all reached statistical significance (p<0.05; 2–factor ANOVA). To our surprise, mice fed the high‐iron diets with low and normal copper levels grew slower and were severely anemic, but the higher copper content normalized growth rate and corrected the anemia. Transferrin saturation, hepatic non‐heme iron content, and hepatic hepcidin mRNA expression were all uniformly elevated in all groups of mice fed the high‐iron diets. We also noted decreased heart copper content and cardiac hypertrophy in the mice fed the high‐iron diet with low and normal copper, but the high‐copper diet corrected this defect. Moreover, hepatic copper content was lower and serum ceruloplasmin activity was depressed in the high‐iron fed mice with the low and normal copper content, but both were restored to control levels in the group fed the high‐iron diet with high copper. These findings suggested that the high dietary iron intake with low and normal copper caused severe copper‐deficiency anemia. We postulated that the high‐iron content blocked intestinal copper absorption, which then resulted in systemic copper deficiency. We thus tested this hypothesis by performing in vivo intestinal copper absorption studies. Mice were again fed the same diets for 4 weeks, fasted overnight and 64Cu was then administered to the mice by oral gavage. Mice were immediately given ad libitum access to the same diets and then sacrificed ~8 hours later. This time point ensured that no copper was excreted in the feces since transit time in mice is ~11 hours. This is important since copper can be excreted in the bile via the feces. Copper absorption was calculated as a ratio of counts in the carcass (minus the GI tract) to total counts administered by gavage × 100. Results showed that mice fed the low copper, adequate‐iron diet had higher copper absorption (>60% of administered dose). Mice fed the low‐copper diet with high iron, however, failed to upregulate copper absorption. Further, although mice fed the adequate copper, high‐iron diet, suffered from copper‐deficiency anemia, they did not have the ability to upregulate copper absorption. Additionally, 64Cu accumulation in all tissues tested was significantly lower than in the low‐copper, adequate‐iron fed group. Collectively, these data then suggest that high‐body iron levels impair intestinal copper absorption and perturb copper distribution, providing further evidence of physiologically relevant iron‐copper interactions in mammals.Support or Funding InformationNIH grant 1R01 DK074867 (to J.F.C.) Diet iron and copper concentration: For the iron and copper interaction study, the diets were fabricated based on the AIN‐93G formulation (Dyets Inc., Bethlehem). Adequate, and high‐iron diets contained 79, 8820 ppm iron, respectively. Low, adequate, and high‐copper diets contained 0.9, 8, 183 ppm copper, respectively. Each 3 iron and copper contents were used to formulate the 9 different diets. The diets were otherwise identical. Fe(ppm) Cu(ppm) FeACuD 93.7 0.92 FeACuA 71.9 8.96 FeACuE 71.8 183 FeECuD 9036 0.94 FeECuA 8707 9.18 FeECuE 8718 184