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© 2004 The American Society for Nutritional Sciences J. Nutr. 134:1953-1957, August 2004

Nutrient Interactions and Toxicity

Copper Deficiency Reduces Iron Absorption and Biological Half-Life in Male Rats1,2

Philip G. Reeves3 and Lana C. S. DeMars

U.S. Department of Agriculture, ARS, Grand Forks Human Nutrition Research Center, Grand Forks, ND 58203

3To whom correspondence should be addressed. E-mail: preeves{at}gfhnrc.ars.usda.gov.


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
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 59Fe 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 59Fe. Retained 59Fe was monitored for 10 d by WBC; then rats were killed and 59Fe 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 59Fe 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) 59Fe in the feces in a 24-h period than the CuA rats. Also, 59Fe in the duodenal mucosa of CuD rats was ~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.

KEY WORDS: • copper • iron • absorption • biological half-life • rats

Dietary copper (Cu) is required for the efficient utilization of dietary iron (Fe). Signs of Fe deficiency anemia appear within 2 wk of first feeding weanling rats a diet deficient in Cu (1). These signs include, among others, low blood hemoglobin, low hematocrit, and low RBC count (2). It seems logical to suggest that part of the problem is that the deficiency inhibits Fe absorption from the gut. Indeed, the Cu-dependent intestinal ferroxidase, hephaestin, is required for Fe absorption (3,4). However, this expected mechanism for the initiation of Cu deficiency anemia has not been consistently borne out by experimentation. Although earlier studies showed that Cu deficiency reduces Fe absorption in pigs (5,6) and rats (7), another report (8) showed that female but not male offspring of Cu-deficient rats had lower Fe absorption than offspring of Cu-adequate rats. A more recent report (9) suggests that Fe absorption is actually enhanced in Cu-deficient female rats. In an attempt to find an unequivocal solution to this controversy, we designed an experiment to determine Fe absorption in Cu-deficient rats by using radioactive iron, 59Fe, and the whole-body counting (WBC)4 technique.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 LITERATURE CITED
 
This study was approved by the Animal Use Committee of the USDA-ARS, Grand Forks Human Nutrition Research Center and followed the guidelines of the National Institutes of Health for the experimental use of laboratory animals (10).

The study design consisted of 2 experimental groups, Cu adequate (CuA) and Cu deficient (CuD). The diets were based on the AIN-93G formulation (11,12) and contained 5.0 mg Cu/kg in the CuA group and <0.3 mg Cu/kg in the CuD group. This diet contained Fe in the form of ferric citrate. Male Sprague-Dawley rats [n = 90; Strain: SAS:VAF (SD), Charles River/Sasco] at 3 wk of age were randomly divided into 2 groups of 45 rats each and fed 1 of the 2 diets. Rats were housed in a temperature- (22°C) and humidity-controlled (50%) room with a 12-h light:dark cycle (lights on 0600–1800 h), and body weights were recorded weekly. At d 0, 4, 7, 14, 21, 28, and 35, the effects of CuD on various parameters were determined in whole blood collected from 5 rats in each group.

At d 7 and 25, 5 rats from each group were deprived of food from 0800 to 1800 h. When the lights went out, rats were fed 1.0 g of their respective diets that had been exogenously labeled with 59Fe. The labeling procedure consisted of suspending a specified amount of diet in deionized water (1:1; wt/v) and blending. After the addition of 37 kBq (1.0 µCi) of 59Fe, as ferric chloride, per gram of diet, the suspension was mixed and allowed to equilibrate overnight at 4°C. The mixture was frozen at –80°C and lyophilized. The dried diet was thoroughly mixed and a 1.0-g meal was offered to each rat in an acid-washed glass jar. After the rats had consumed the meal (<30 min), the amount of 59Fe in each was determined by WBC. The whole-body counter design and general technique were described previously (13). WBC was repeated at 12 h, and each day thereafter for 9–10 d. During the last 48 h of the WBC period, feces were collected for the determination of 59Fe excretion. These data were expressed as percentages of the final whole-body count.

At the end of the WBC period, the rats were killed without being deprived of food. Whole blood was collected from the abdominal aorta and immediately analyzed with a Cell-Dyn 3500 automated hematology cell counter (Abbott Laboratories) to assess the effects of Cu deficiency on erythrocyte (RBC) count, hemoglobin concentration, hematocrit, mean corpuscular volume (MCV), red cell distribution width (RDW), and platelet number. To assess Cu status, a second sample of blood was collected at each kill, and the serum separated for the analysis of ceruloplasmin (Cp) amine oxidase activity (14). Duodenal mucosa, liver, and spleen were collected for the analysis of 59Fe content.

Log-linear regressions analyses of the percentage of isotope retention vs. time in days were run on the linear part of the curves. For the trial beginning at d 7, this was for d > 3 and for the trial beginning at d 25, it was for d > 4. The y-intercept from each regression was used as an estimate of the percentage absorption. The biological half-life (BHL) of 59Fe was calculated as –ln(2)/slope. Because the distributions of the BHL were skewed, these data were ln-transformed before statistical analyses were performed. Statistical differences between groups for absorption and BHL were verified by using Student’s t statistic. A two-way ANOVA was used to identify differences in means for the distribution of whole-body 59Fe and fecal excretion of the isotope, and significance was set at P < 0.05. Where there was a significant interaction, Tukey’s contrast analysis was used to discern differences between means.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
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DISCUSSION
 LITERATURE CITED
 
Weight gains in both CuA and CuD rats were similar until ~wk 4 of the experiment (Fig. 1). At that point, rats in the CuD group began to gain less (P < 0.05) weight than the CuA rats, and continued to weigh less until the end of the experiment.



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  		FIGURE 1 Copper deficiency reduces body weight in rats. Weanling male rats were fed semipurified diets with (CuA) and without (CuD) adequate Cu. Values are the means ± SEM, n = 5. *Different from CuD, P ≤ 0.05.

 
When 59Fe-labeled diet was fed to rats after 7 d, the amount of label absorbed by the CuD group tended to be lower than that for the CuA group [63 ± 2% vs. 69 ± 3% (mean ± SEM), respectively] (P > 0.15) (Fig. 2). However, a determination of BHL showed that CuD rats lost 59Fe significantly (P < 0.02) faster than CuA rats [geometric mean (–SEM, +SEM); 75(62,91) d vs. 175(156,195) d, respectively]. Serum Cp activity, an indicator of Cu status, was lower (P < 0.001) in CuD than CuA rats at the beginning of this absorption test period (d 7) (Fig. 3), indicating that Cu deficiency was established. However, signs of Fe deficiency anemia had not developed at this time; hematocrit and hemoglobin did not differ (P > 0.16) between groups (Fig. 4). Nonetheless, near the end of this absorption period (d 14), rats fed the CuD diet were showing signs of Fe deficiency in that blood hemoglobin, hematocrit, and RBC number were lower (P < 0.001) than in rats fed the CuA diet (Fig. 4).



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  		FIGURE 2 Copper deficiency for 7 d decreases the BHL but not absorption of 59Fe in male rats. Values are means ± SEM, n = 5. The linear portion of each curve (·······) was extrapolated to d 0 (shown here for illustrative purposes only) and this value was used to estimate the amount of 59Fe absorbed (see text). Absorption did not differ between CuA and CuD rats (P > 0.10); however, the BHL of 59Fe in the CuD rats was less than half (P < 0.02) that for the CuA rats.

 


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  		FIGURE 3 Copper deficiency reduces the activity of Cp in the serum of male rats. Values are means ± SEM, n = 5. Some of the SEM bars are hidden in the symbols. *Different from CuD, P < 0.001.

 


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  		FIGURE 4 Copper deficiency affects various blood parameters of male rats. Values are means ± SEM, n = 5. *Different from CuD, P ≤ 0.02.

 
When 59Fe-labeled diet was fed to rats after 25 d, the percentage absorption of Fe in the CuD rats was only about half that of the CuA rats [33 ± 2% vs. 60 ± 3%, respectively (P < 0.001)] (Fig. 5). At this time, the BHL of 59Fe in the CuD rats was again shorter (P < 0.02) than that for CuA rats [33(23,49) d vs. 157(148,166) d, respectively].



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  		FIGURE 5 Copper deficiency for 25 d decreases absorption and the BHL of 59Fe in male rats. Values are means ± SEM, n = 5. The linear portion of each curve (·······) was extrapolated to d 0 (shown here for illustrative purposes only) and this value was used to estimate the amount of 59Fe absorbed (see text). Absorption differed between CuA and CuD rats, P < 0.001. The BHL was also lower (P < 0.02) in CuD rats than in CuA rats.

 
To determine where the loss of body Fe might be occurring, fecal material was collected during the last 2 d of the WBC period and assayed for 59Fe. At d 16 of the study in which labeled diets had been fed on d 7, the CuD rats excreted 4 times (P < 0.008) more label in a 24-h period than the CuA rats (Table 1). Although all rats were excreting more of the label into the feces by d 34, the CuD rats were excreting an even higher percentage of the label than the CuA rats [significant interaction (P < 0.012) between diet and time on experiment].


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  		TABLE 1 Distribution of whole-body 59Fe in whole blood, blood fractions, liver, spleen, duodenal mucosa, and 24-h fecal collections in CuD and CuA male rats that were administered the label on d 7 or 251, 2

 
Cp activity of the CuD rats was lower (P < 0.001) than that of the CuA rats as early as d 4 after first consuming the Cu deficient diet (Fig. 3). From that point to the end of the experiment, Cp activity in the CuD rats remained lower (P < 0.001) than that of the CuA rats.

The distribution of whole-body 59Fe in blood components and organs was affected by CuD and time on experiment (Table 1). The percentage of the label distributed to whole blood was less in CuD rats than CuA rats during both trials; however, the difference between groups was much greater (P < 0.008) during the trial that began on d 25 than on d 7. Because most of the Fe in blood is contained in the RBC, blood cell distribution patterns of 59Fe were similar to those of whole blood. On the other hand, distribution of 59Fe into plasma was not affected by dietary Cu, but it was much higher (P < 0.001) in the trial started on d 7 of the experiment than in the one started on d 25.

The distribution of 59Fe into the liver was higher in CuD rats than CuA rats and the magnitude depended upon how long the rats were kept under experiment conditions. Expressed as a percentage of whole-body 59Fe, the amount of label retained in the liver was much higher (P < 0.001) when administered at d 25 of the experiment than when administered at d 7. Spleen 59Fe was higher (P < 0.001) in CuD rats than CuA rats, and the value at d 25 was slightly lower (P < 0.02) than at d 7 of Cu depletion. There was a larger percentage of whole-body 59Fe in the mucosa of CuD rats than in the CuA rats at both periods, but there was no effect of length of exposure to Cu deficiency. The sum of the percentages of whole blood and organs equaled only ~59% of the total in the whole animal, suggesting that the 59Fe was distributing to other tissues and organs not assayed in this study, such as muscle, kidney, and bone. However, the measured components accounted for less (P < 0.002) label in the CuD rats than in the CuA rats. Although a decrease in whole blood 59Fe accompanied an increase in liver 59Fe in the CuD rats, the increase in liver was not enough to account for the overall decrease in whole blood.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
The data from this experiment show unequivocally that Cu deficiency reduces Fe absorption in male rats. At 25 d after first feeding the deficient diet, Fe absorption in CuD rats was 50% of that in the CuA rats. In previous studies, it was shown that Fe absorption in pigs was reduced by Cu deficiency (6), but mixed results were obtained when rats were used as the experimental model. Work by Chase et al. (15) suggested that rats with low Cu status absorbed less Fe than those with adequate status. Later studies by Cohen et al. (8) found that Fe absorption was significantly reduced only in female offspring of Cu-deficient dams. By using an intestinal/vascular perfusion technique in rats, Coppen and Davies (7) showed that Fe uptake from the luminal media into the enterocytes was not affected by Cu deficiency but the rate of 59Fe transfer to the vascular perfusate was significantly reduced.

Recent reports also showed that Cu affects Fe uptake in different types of cell culture models. Yu and Wessling-Resnick (16) showed that Fe uptake was reduced in Cu-deficient HeLa cells, and that Cu supplements in the media of Caco-2 cells stimulated Fe uptake (17). However, Zerounian and Linder (18) treated Caco-2 cells with triethylenetetraamine to lower the cellular concentration of Cu and found an increase in Fe uptake.

Despite the negative effects that low Cu status has on intestinal Fe absorption and cellular Fe uptake, Thomas and Oates (9) observed that Cu deficiency in female rats increased Fe absorption. These results were opposite to those found for male rats in our study. It is not apparent why the 2 sexes should respond so differently to Cu deficiency, but previous observations showed that the anemia induced by Cu deficiency was less pronounced in female than in male rats (19,20). In this regard, after 8 wk, hematocrits in the study of Thomas and Oates were 0.33 and 0.41 for CuD and CuA, respectively, suggesting only a mild anemia. In our study, hematocrits were 0.24 and 0.40 in rats with hemoglobin concentrations of 80 and 130 g/L, respectively, showing a rather severe anemia in male rats even after only 25 d of consuming the CuD diet. Although their diets were similar to ours, the actual Cu concentrations of their diets are unknown.

Some differences in the design of the 2 absorption procedures also might be important. Thomas and Oates (9) fed an AIN-93G-based diet with 70 mg Fe/kg as ferric citrate and administered 59FeSO4 as a gavage in a HEPES/NaCl buffer containing ascorbic acid. They expressed absorption as a percentage of the dose that remained in the body after 5 d. On the other hand, we administered 59FeCl3 as part of a diet containing 35 mg Fe/kg as ferric citrate, which the rats had consumed throughout the experiment. Rats do not require ascorbic acid; thus, none was added to our diets (21). Then, each day for 10 d, we determined the amount of 59Fe remaining in the body by WBC. Plotting the percentage of the dose remaining each day on a log scale and extrapolating the linear portion of the curve back to zero time gave us an estimate of the amount of 59Fe initially absorbed. This procedure provided a more accurate estimate of apparent absorption than a single-point determination. If these differences in Fe absorption between male and female rats are real, it would be interesting to determine how sexual characteristics affect Cu regulation of Fe absorption.

The results of the present study, showing a 50% reduction in Fe absorption in CuD rats, suggest that at least part of the effects on anemia arises because of inefficient transfer of Fe from the enterocyte to the blood. However, other studies suggest that Cu deficiency causes a redistribution of Fe in the body organs; Fe in whole blood is lower, but it is higher in liver. A study by Failla and Seidel (22) compared the effects of dietary starch and fructose on the total body content of various minerals. They showed that total body Cu was 50% lower in CuD rats than CuA rats, but there was no change in total body Fe, regardless of carbohydrate source. Blood hemoglobin concentrations and liver Fe were not measured in this study; however, based on a hematocrit of 0.31 in 1 CuD group compared with 0.44 in CuA rats, we contend that anemia was evident in CuD rats that had no significant change in total body Fe.

The mechanism(s) by which Cu affects Fe absorption have not been elucidated completely, but recent evidence suggests that a Cu-dependent intestinal ferroxidase, hephaestin (Hp), is involved in the transfer of Fe from the enterocytes to the blood (3,4,23,24). However, Cu deficiency anemia is not completely dependent on intestinal Fe absorption because other studies showed that intramuscular administration of Fe to CuD pigs did not completely cure anemia (25). Although Cp is important for the release of Fe from the reticuloendothelial system and liver cells, some aceruloplasminemic strains of mice are only mildly or not at all anemic (26). A mutant strain of mice, called sla, lacks a functional Hp gene and cannot absorb Fe, which results in anemia; however, if supplied with parenteral Fe, their anemia is prevented (27). Together, these findings suggest that some other Cu-dependent factor is responsible for normal systemic Fe metabolism related to RBC and hemoglobin formation. Is the initial effect at the intestinal level at which Fe absorption is directly affected by the absence of Cu, or does Cu deficiency reduce blood cell formation, which in turn, reduces the need for Fe? The latter might then generate a signal that shuts down intestinal Fe absorption. Indeed, in the current study, there was a greater reduction in Fe absorption after the Cu-deficient rats had developed anemia than before.

In general, our results showing the effects of Cu deficiency on RBC production and hemoglobin concentration are similar to those reported previously (1,2). Johnson and Saari (1) found that serum Cp activity was low in weanling rats at the start of their experiment. Over time, Cp activity gradually increased in the CuA rats but not in the CuD rats. The results of our experiment were somewhat different. Cp activity at the start of the experiment was at a modest level, and it doubled in the CuA group over the next 5 wk of the experiment. On the other hand, Cp activity in the CuD rats declined sharply to almost minimal levels at d 4, and never recovered throughout the remainder of the study. Evans and Abraham (28) measured Cp activity in rats as affected by CuD over time and had results similar to ours. Prohaska and Lukasewycz (29) found that serum Cp activity of mice was radically reduced after only 3 d of CuD treatment. However, they did not discuss the Cp activity of CuA mice over the same period.

To our knowledge, the data presented in this report are the first to show that systemic Fe is turned over more quickly in CuD rats than CuA rats. The BHL of dietary 59Fe in the CuD rats was 30–50% that in CuA rats. We have preliminary evidence that this phenomenon is real, because the BHL of 59Fe injected intraperitoneally with the isotope also was less (P < 0.03) in CuD rats than in CuA rats [geometric mean (range); 122(121–123) d vs. 164(163–165) d, n = 2, respectively]. The normal loss of body Fe usually takes place very slowly; for the most part, it occurs through the feces from bile secretions and desquamated mucosal cells. Indeed, during both periods of administration in the current study, the loss of 59Fe seemed to be through the feces with up to 5 times higher excretion in CuD rats than in adequate rats. How Cu deficiency might increase the excretion of Fe through this route is not understood.

In summary, this study showed that Cu deficiency in male rats does indeed reduce the absorption of dietary iron and, in addition, increases the turnover of body iron. The results suggest that part of the ensuing effect on Fe deficiency anemia is caused by less Fe entering and remaining in the body. However, Cu controls a number of systemic enzymatic reactions that regulate Fe release from reticuloendothelial system, and possibly those that function in hemoglobin and blood cell formation. The role of Cu in orchestrating these different mechanisms in the regulation of Fe metabolism is the subject of ongoing research.


   ACKNOWLEDGMENTS
 
The authors acknowledge the assistance of Denice Schafer and staff for care of the animals, Jim Lindlauf for preparation of the diet, Brenda Skinner and Matt Soule for assistance with animal kills and blood analyses on the Cell-Dyn, and LuAnn Johnson for the statistical analyses.


   FOOTNOTES
 
1 Presented at Experimental Biology 04, April 2004, Washington, DC [Reeves, P. G. & DeMars, L. (2004) Copper deficiency in rats reduces iron absorption and increases the rate of iron loss from the body]. Back

2 Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. Back

4 Abbreviations used: BHL, biological half-life; Cp, ceruloplasmin; CuA, copper adequate; CuD, copper deficiency/copper deficient; MCV, mean corpuscular volume; RDW, red cell distribution width; WBC, whole-body counting. Back

Manuscript received 22 March 2004. Initial review completed 15 April 2004. Revision accepted 11 May 2004.


   LITERATURE CITED
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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1. Johnson, W. T. & Saari, J. T. (1991) Temporal changes in heart size, hematocrit and erythrocyte membrane protein in copper-deficient rats. Nutr. Res. 11:1403-1414.

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 Am. J. Physiol. Gastrointest. Liver Physiol.Home page
M. Wessling-Resnick
Iron Imports. III. Transfer of iron from the mucosa into circulation
Am J Physiol Gastrointest Liver Physiol, January 1, 2006; 290(1): G1 - G6.
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 Exp. Biol. Med.Home page
P. G. Reeves and L. C. S. DeMars
Repletion of Copper-Deficient Rats with Dietary Copper Restores Duodenal Hephaestin Protein and Iron Absorption
Experimental Biology and Medicine, May 1, 2005; 230(5): 320 - 325.
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 J. Nutr.Home page
P. G. Reeves, L. C. S. DeMars, W. T. Johnson, and H. C. Lukaski
Dietary Copper Deficiency Reduces Iron Absorption and Duodenal Enterocyte Hephaestin Protein in Male and Female Rats
J. Nutr., January 1, 2005; 135(1): 92 - 98.
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