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. 2016 Aug 18;11(8):e0161033.
doi: 10.1371/journal.pone.0161033. eCollection 2016.

High-Iron Consumption Impairs Growth and Causes Copper-Deficiency Anemia in Weanling Sprague-Dawley Rats

Jung-Heun Ha  1 , Caglar Doguer  1 , Xiaoyu Wang  1 , Shireen R Flores  1 , James F Collins  1
Affiliations

High-Iron Consumption Impairs Growth and Causes Copper-Deficiency Anemia in Weanling Sprague-Dawley Rats

Jung-Heun Ha et al. PLoS One. .

Abstract

Iron-copper interactions were described decades ago; however, molecular mechanisms linking the two essential minerals remain largely undefined. Investigations in humans and other mammals noted that copper levels increase in the intestinal mucosa, liver and blood during iron deficiency, tissues all important for iron homeostasis. The current study was undertaken to test the hypothesis that dietary copper influences iron homeostasis during iron deficiency and iron overload. We thus fed weanling, male Sprague-Dawley rats (n = 6-11/group) AIN-93G-based diets containing high (~8800 ppm), adequate (~80) or low (~11) iron in combination with high (~183), adequate (~8) or low (~0.9) copper for 5 weeks. Subsequently, the iron- and copper-related phenotype of the rats was assessed. Rats fed the low-iron diets grew slower than controls, with changes in dietary copper not further influencing growth. Unexpectedly, however, high-iron (HFe) feeding also impaired growth. Furthermore, consumption of the HFe diet caused cardiac hypertrophy, anemia, low serum and tissue copper levels and decreased circulating ceruloplasmin activity. Intriguingly, these physiologic perturbations were prevented by adding extra copper to the HFe diet. Furthermore, higher copper levels in the HFe diet increased serum nonheme iron concentration and transferrin saturation, exacerbated hepatic nonheme iron loading and attenuated splenic nonheme iron accumulation. Moreover, serum erythropoietin levels, and splenic erythroferrone and hepatic hepcidin mRNA levels were altered by the dietary treatments in unanticipated ways, providing insight into how iron and copper influence expression of these hormones. We conclude that high-iron feeding of weanling rats causes systemic copper deficiency, and further, that copper influences the iron-overload phenotype.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. High-iron feeding impaired growth and caused cardiac hypertrophy.
Weanling rats were fed one of 9 diets differing only in iron and copper content for 5 weeks ad libitum. Rats were weighed weekly, and growth rates were calculated (A). Final body weights (B), and liver (C) and heart (D) weights at sacrifice are also shown. Organ weights were normalized by body weight. Values are means ± SDs. Labeled means without a common letter differ (p<0.05). Animal numbers were as follows: LFe/LCu, n = 9; LFe/AdCu and LFe/HCu, n = 6; AdFe/AdCu, n = 11; and all others, n = 10. These same n values apply to all data presented in this manuscript (which will not be repeated in subsequent figure legends). Abbreviations: L, low; Ad, adequate; H, high. 2-way ANOVA factor analysis results are as follows: growth rate (Fe: p<0.0001; Cu: p<0.0001; Fe X Cu: p<0.001); final body weight (Fe: p<0.0001; Cu: p<0.01; Fe X Cu: p<0.001); liver weights (Fe: p<0.0001; Cu: ns; Fe X Cu: p<0.05); and heart weights (Fe: p<0.0001; Cu: p<0.0001; Fe X Cu: p<0.01). ns, not significant.
Fig 2
Fig 2. Consumption of the low- and high-iron diets altered hematological parameters.
Hemoglobin (Hb) (A) and hematocrit (Hct) (B) were determined from whole blood collected from experimental animals at sacrifice. Serum nonheme iron (C), serum transferrin saturation (D) and total iron-binding capacity (TIBC) (E) were also quantified. Labeled means without a common letter differ (p<0.05). n values and abbreviations used are the same as in Fig 1. The Box-and-Whisker plots indicate the following: the minimum value (the lower whisker), the lower quartile, the median, the upper quartile and the maximum value (the upper whisker). 2-way ANOVA factor analysis results are as follows: hemoglobin (Fe: p<0.0001; Cu: p<0.0001; Fe X Cu: p<0.0001); hematocrit (Fe: p<0.0001; Cu: p<0.0001; Fe X Cu: p<0.0001); serum nonheme iron (Fe: p<0.0001; Cu: p<0.001; Fe X Cu: p<0.0001); serum transferrin saturation (Fe: p<0.0001; Cu: p<0.0001; Fe X Cu: p<0.001) and TIBC (Fe: p<0.0001; Cu: ns; Fe X Cu: p<0.001). ns, not significant.
Fig 3
Fig 3. Renal Epo and splenic Erfe levels increased in rats consuming the LFe/LCu diet.
Renal Epo mRNA (A) and serum Epo protein levels (B) were assessed in experimental rats. The Pearson product-moment correlation coefficient (r) comparing these 2 parameters is noted in the inset of panel B (p<0.0001). Splenic Erfe mRNA expression was quantified by qRT-PCR (C) and splenic nonheme iron levels (D) were measured using a commonly used technique. Labeled means without a common letter differ (p<0.05). Values are means ± SDs. n values and abbreviations used are the same as in Fig 1. Data for Epo mRNA and protein expression and splenic nonheme iron concentration were log10 transformed prior to running statistical analyses due to large variations in values. For ease of interpretation, we have, however, presented the non-transformed data in the figure. 2-way ANOVA factor analysis results are as follows: renal Epo mRNA (Fe: p<0.0001; Cu: p<0.0001; Fe X Cu: p<0.0001); serum Epo protein (Fe: p<0.0001; Cu: p<0.0001; Fe X Cu: p<0.0001); splenic Erfe (Fe: p<0.0001; Cu: p<0.001; Fe X Cu: p<0.0001); and splenic nonheme iron (Fe: p<0.0001; Cu: p<0.01; Fe X Cu: p<0.01).
Fig 4
Fig 4. Extra copper in the HFe diets did not influence Hepc mRNA expression but it accentuated hepatic nonheme iron loading.
Hepc mRNA expression was quantified in experimental rats (A), and the relationship between Hepc mRNA expression (log10) and Tf saturation was estimated by calculating Pearson product-moment correlation (B). The line of best fit is shown along with the correlation coefficient (r) (p<0.0001). Hepatic total (C, left side) and nonheme (C, right side) iron was also measured. Note that the 2 halves of panel C are mirror images with respect to the order of the experimental groups. Correlations were also calculated between Hepc mRNA expression (log10) and liver iron levels (log10) (r values are shown as insets) (C). Labeled means without a common letter differ (p<0.05). Values are means ± SDs. n values and abbreviations used are the same as in Fig 1. Data for Hepc mRNA and hepatic total and nonheme iron concentrations were log10 transformed prior to running statistical analysis due to large variations in values. For ease of interpretation, we have, however, presented the non-transformed data in the figure. a.u., arbitrary units. 2-way ANOVA factor analysis results are as follows: Hepc (Fe: p<0.0001; Cu: ns; Fe X Cu: p<0.001); total liver iron (Fe: p<0.0001; Cu: ns; Fe X Cu: p<0.01); and hepatic nonheme iron (Fe: p<0.0001; Cu: p<0.01; Fe X Cu: p<0.01). ns, not significant.
Fig 5
Fig 5. High-iron feeding resulted in severe tissue copper depletion and reduced Cp activity.
The copper concentration in liver (A), serum (B), heart (C) and bone (D) was determined by ICP-MS. Cp (i.e. amine oxidase) activity was also measured in serum samples (E). The correlation between Cp activity and liver copper concentrations (log10) was calculated using Pearson product-moment correlation analysis (F). The line of best fit is shown along with the correlation coefficient (r) (p<0.0001). Labeled means without a common letter differ (p<0.05). n values and abbreviations used are the same as in Fig 1. The Box-and-Whisker plots indicate the following: the minimum value (the lower whisker), the lower quartile, the median, the upper quartile and the maximum value (the upper whisker). 2-way ANOVA factor analysis results are as follows: liver copper (Fe: p<0.0001; Cu: p<0.0001; Fe X Cu: p<0.0001); serum copper (Fe: p<0.05; Cu: p<0.0001; Fe X Cu: p<0.01); heart copper (Fe: p<0.0001; Cu: p<0.0001; Fe X Cu: p<0.0001); bone copper (Fe: p<0.01; Cu: p<0.0001; Fe X Cu: ns); and Cp activity (Fe: p<0.0001; Cu: p<0.0001; Fe X Cu: p<0.0001).
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