Determinants of copper-deficiency anemia in rats

Indicators of copper and iron metabolism were studied in pregnant rats and their 90-d-old offspring fed copper-sufficient or copper-deficient diets containing marginal or adequate levels of iron from the beginning of pregnancy until the offspring were 90 d of age. Offspring had more severe signs of copper deficiency (including anemia, hypertrophy of the heart, decreased activity of ferroxidase I and II, depression of growth and death) than the dams. In both dams and offspring, copper deficiency resulted in anemia when dietary iron was marginal but not when it was adequate. Liver iron was elevated in copper-deficient male offspring, but not in female offspring. Anemia and growth retardation were more pronounced in copper-deficient males than in females, despite similarly low levels of ferroxidase I and II. Iron absorption was reduced by copper deficiency only in female offspring. Activity of 59Fe in various tissues 6 or 48 h after gavage did not reveal any other effect of copper deficiency on iron metabolism. Thus age at the time copper-deficient diets were introduced, sex and dietary iron strongly influence the effect of copper deficiency.     

Influence of iron and the sex of rats on hematological, biochemical and immunological changes during copper deficiency

The influence of dietary iron and the sex of rats on suppressed lymphocyte functions caused by copper deficiency was examined. Male and female weanling Lewis rats were fed two concentrations of copper (0.6 or 5.6 micrograms Cu/g diet) and iron (50 or 300 micrograms Fe/g diet) for 42 d. Regardless of dietary iron concentrations, male and female rats consuming low copper diets had lower serum ceruloplasmin activity and serum and liver copper concentrations than those fed the high copper diet. However, hemoglobin and hematocrit levels were higher in copper-deficient females than in copper-deficient males and were unaffected by copper deficiency in females fed the high iron diet. Copper-deficient females also had higher serum and liver iron concentrations than copper-deficient males. Proliferation of concanavalin A (Con A)-stimulated spleen lymphoid cells (SLC) was suppressed in copper-deficient males and females, but the suppression was less in the females. Thus, the primary cause of suppressed SLC proliferation in copper-deficient rats is poor copper status; poor iron status induced by copper deficiency had little influence on proliferation.     

Plasma diamine oxidase activity is greater in copper-adequate than copper-marginal or copper-deficient rats

The object of this study was to determine whether serum diamine oxidase activity could distinguish among adequate, marginal and deficient copper status in rats. Male weanling Sprague-Dawley rats (n = 21) were randomly assigned to one of three dietary regimens, with copper concentrations of 0.52, 1.73 and 6.7 mg/kg diet. On completion of the study, body weights were significantly different among dietary groups, with copper-marginal rats displaying the highest mean weight and copper-deficient rats the lowest. Copper-deficient rats ate significantly less food than the other two groups. Rats fed the three diets had significantly different liver copper concentrations. Liver and heart superoxide dismutase and cytochrome c oxidase activities, and plasma ceruloplasmin and erythrocyte superoxide dismutase activities were significantly lower in the copper-deficient rats than in the other two groups. Plasma diamine oxidase activity was lower in both copper-deficient (0.18 +/- 0.11 U/L) and marginal (0.21 +/- 0.11 U/L) rats compared with copper-adequate rats (3.35 +/- 0.28 U/L). Of the biochemical indices measured, only liver copper concentration (-20%) and plasma diamine oxidase activity (-94%) differed between rats fed copper-marginal and copper-adequate diets. Plasma diamine oxidase activity, therefore, may be a sensitive functional biomarker of suboptimal copper status.     

Development of anemia in copper-deficient rats fed high levels of dietary iron and sucrose

The effects of dietary carbohydrate and iron on the development of copper deficiency were examined. Male Sprague-Dawley rats (n = 48) were limit-fed one of eight diets in a 2 X 2 X 2 factorial design for 19 d. Two levels of copper (0.85 or 8.6 micrograms Cu/g diet) and iron (54 or 226 micrograms Fe/g diet) and two types of carbohydrate (sucrose or cornstarch, 65.3%) were fed. Compared with control rats, copper-deficient rats had lower hematocrits, lower ceruloplasmin levels, lower tissue levels of copper and increased hepatic iron levels. Copper-deficient rats fed sucrose had significantly lower hematocrits, lower apparent absorption of copper, lower liver iron levels and higher plasma triglyceride levels than copper-deficient rats fed cornstarch. Copper-deficient rats fed sucrose with 226 micrograms Fe/g diet had hematocrit levels that were 15% lower than all other copper-deficient levels and 23% lower than control levels. Tissue levels of copper among copper-deficient rats were not affected by the type of carbohydrate or by the level of dietary iron. These data indicate that both high iron and sucrose can affect the development of the copper deficiency.     

Level of dietary iron,not type of dietary fat,is hyperlipidemic in copper-deficient rats

OBJECTIVE: This study was conducted to determine whether high dietary iron will negate the protective effect of unsaturated fat against hyperlipidemia. METHODS: Forty-eight weanling, male Sprague Dawley rats were randomly assigned to eight dietary groups differing in the levels of copper and iron and type of dietary fat (saturated or unsaturated). The diets were either deficient (0.6 microg Cu/g) or adequate (6.8 microg Cu/g) copper and either adequate (53 microg Fe/g) or high (506 microg Fe/g) iron. All diets contained starch as the sole source of dietary carbohydrate. RESULTS: Regardless of the type of dietary fat, three copper-deficient rats fed the high levels of dietary iron died prematurely due to ruptured hearts. Surviving rats belonging to the copper deficiency and high-dietary iron regimen developed severe anemia, enlarged hearts and livers, and exhibited the highest levels of liver iron. These rats also developed hypercholesterolemia. Triglycerides were elevated by the consumption of high iron diets. CONCLUSION: Data show that levels of dietary iron, not the type of dietary fat, are potential inducers of hypertriglyceridemia. Data also show that the combination of high iron intake and dietary copper deficiency is responsible for elevating blood cholesterol.     

Influence of sucrose and starch on the development of anemia in copper- and iron-deficient rats

The purpose of this investigation was to compare the effects of sucrose and starch on the development of copper and iron deficiency. Male Sprague-Dawley rats (n = 48) were fed one of eight diets in a 2 X 2 X 2 factorial design for 24 d. Two levels of copper (deficient, 0.7 microgram/g, or adequate, 8.3 micrograms/g) and iron (deficient, 8.3 micrograms/g, or adequate, 50 micrograms/g), and two types of carbohydrate (sucrose or starch, 62% of the diet) were fed. Copper-deficient rats had significantly lower hematocrit, hemoglobin and tibia iron levels and depressed copper and iron absorption when fed sucrose instead of starch. The apparent absorption of copper, but not iron, was significantly lower when rats deficient in both copper and iron were fed sucrose rather than starch. Iron-deficient rats fed sucrose apparently absorbed significantly more iron than those fed starch; however, sucrose did not significantly improve hematocrit and hemoglobin levels. The metabolism of copper and iron by rats fed diets adequate in these nutrients was not affected by the type of dietary carbohydrate. These data indicate that the type of dietary carbohydrate alters both copper and iron metabolism, particularly in copper-deficient rats.     

Copper uptake and retention in liver parenchymal cells isolated from nutritionally copper-deficient rats

Copper uptake and retention were studied in primary cultures of liver parenchymal cells isolated from copper-deficient rats. Male Sprague-Dawley rats were fed a copper-deficient diet (less than 1 mg Cu/kg) for 10 wk. Copper-deficient rats were characterized by low copper concentrations in plasma and liver, anemia, low plasma ceruloplasmin oxidase activity and increased 64Cu whole-body retention. Freshly isolated liver parenchymal cells from copper-deficient rats showed a higher 64Cu influx, which was associated with a higher apparent Vmax of 45 +/- 4 pmol Cu.mg protein-1.min-1 as compared with 30 +/- 3 pmol Cu.mg protein-1.min-1 for cells isolated from copper-sufficient rats. No significant difference in the apparent Km (approximately 30 mumol/L) was observed. Relative 64Cu efflux from cells from copper-deficient rats was significantly smaller than the efflux from cells from copper-sufficient rats after prelabeling as determined by 2-h efflux experiments. Analysis of the medium after efflux from cells from copper-deficient rats showed elevated protein-associated 64Cu, suggesting a higher incorporation of radioactive copper during metalloprotein synthesis. Effects of copper deficiency persist in primary cultures of parenchymal cells derived from copper-deficient rats, and short-term cultures of these cells offer a prospect for the study of cell biological aspects of the metabolic adaptation of the liver to copper deficiency.     

Lipid alterations in the liver and serum of rats in histidine-excess and copper deficiency

To obtain further information on lipid metabolism in the histidine-excess and copper-deficiency, rats were fed basal, histidine-excess (the addition of 50 g L-histidine/kg diet) or copper-deficient diets for 0, 7, 21 and 42 d ad libitum. Liver triacylglycerol accumulated and the serum triacylglycerol level decreased after feeding of the histidine-excess diet for 21 or 42 d, but not after feeding of the copper-deficient diet. Serum cholesterol level increased in rats fed the histidine-excess diet for 7, 21 and 42 d, but not in rats fed the copper-deficient diet. Copper content in the liver and serum significantly decreased in rats fed the histidine-excess diet. Copper content in the liver and serum was markedly decreased in rats fed the copper-deficient diet. Liver zinc content was constant, but the serum zinc level decreased in rats fed the histidine-excess diet. Feeding of the copper-deficient diet hardly affected zinc content in the liver and serum. Urinary copper and zinc increased in rats fed the histidine-excess diet, and decreased or showed a decreasing tendency in rats fed the copper-deficient diet. Overall results indicated that feeding the histidine-excess diet caused copper deficiency, whereas hypercholesterolemia was not shown in rats fed the copper-deficient diet although the livers of rats fed the copper-deficient diet contained less copper than those of rats fed the histidine-excess diet. Thus, the responses on liver triacylglycerol and serum cholesterol to copper deficiency induced by the feeding of a histidine-excess diet are different from those to copper deficiency induced by feeding of a copper-deficient diet.     

Total body content of copper and other essential metals in rats fed fructose or starch

The effects of dietary carbohydrate and copper on whole body levels of copper and other essential minerals were investigated. Weaned male rats were pair-fed diets containing 62% fructose or cornstarch with either adequate (6–7 mg/kg) or deficient (0.5–0.7 mg/kg) copper (Cu) for 32–33 days. Recommended levels of other minerals were present in all diets. Total copper in whole body was approximately 2-fold higher in rats fed copper-supplemented diets than copper-deficient diets and was independent of the type of dietary carbohydrate. Whole body levels of zinc, iron, manganese and magnesium were independent of dietary copper and carbohydrate. Selenium content of rats fed copper-deficient diet with fructose was 35% higher than in those fed copper-adequate diet with fructose. Rats fed the copper-deficient diet containing starch had a whole body calcium level 17–23% lower than that of the other dietary groups. These results demonstrate that the copper-carbohydrate interaction is not a result of carbohydrate-dependent effects on the retention of dietary copper and other essential minerals.     

Level of Dietary Iron,Not Type of Dietary Fat,is Hyperlipidemic in Copper-Deficient Rats

Objective: This study was conducted to determine whether high dietary iron will negate the protective effect of unsaturated fat against hyperlipidemia.

Methods: Forty-eight weanling, male Sprague Dawley rats were randomly assigned to eight dietary groups differing in the levels of copper and iron and type of dietary fat (saturated or unsaturated). The diets were either deficient (0.6 μg Cu/g) or adequate (6.8 μg Cu/g) copper and either adequate (53 μg Fe/g) or high (506 μg Fe/g) iron. All diets contained starch as the sole source of dietary carbohydrate.

Results: Regardless of the type of dietary fat, three copper-deficient rats fed the high levels of dietary iron died prematurely due to ruptured hearts. Surviving rats belonging to the copper deficiency and high-dietary iron regimen developed severe anemia, enlarged hearts and livers, and exhibited the highest levels of liver iron. These rats also developed hypercholesterolemia. Triglycerides were elevated by the consumption of high iron diets.

Conclusion: Data show that levels of dietary iron, not the type of dietary fat, are potential inducers of hypertriglyceridemia. Data also show that the combination of high iron intake and dietary copper deficiency is responsible for elevating blood cholesterol.     

Interaction of dietary carbohydrate, ascorbic acid and copper with the development of copper deficiency in rats

The effects of dietary carbohydrate and ascorbic acid on the development of copper deficiency were investigated. Male Sprague-Dawley rats (n = 48) were fed one of eight diets in a 2 X 2 X 2 factorial design for 21 d. These diets varied in copper (1.11 or 8.96 micrograms Cu/g diet), carbohydrate (sucrose or cornstarch, 62.3%) and ascorbic acid (0 or 1%). Compared to controls, copper-deficient rats had lower hematocrit and ceruloplasmin levels, lower levels of copper and iron in several tissues, higher heart weights and lower spleen weights. During copper deficiency, liver iron levels were higher than control levels when cornstarch, but not sucrose, was the carbohydrate source, while liver and gastrointestinal tract weights were higher with sucrose compared to cornstarch. Copper-deficient rats fed ascorbic acid had significantly (P less than 0.05) lower hematocrits when fed sucrose compared to starch [29.6 +/- 1.2 vs. 36.8 +/- 1.2 g/dl (mean +/- SEM), respectively]. In copper-deficient rats, sucrose tended to lower the apparent absorption of copper compared to cornstarch, while ascorbic acid reduced the apparent absorption of iron. Thus, sucrose and ascorbic acid appeared to reduce hematocrit levels through effects on mineral absorption.     

Beta-carotene 15,15'-dioxygenase activity is responsive to copper and iron concentrations in rat small intestine

OBJECTIVE: Previous in vitro studies have suggested that beta-carotene 15,15'-dioxygenase is an iron-dependent enzyme. However, in vivo, it is difficult to alter iron tissue concentration by varying dietary iron because of homeostatic control. On the other hand, an interaction between iron and copper has been shown, i.e., copper-deficiency results in an increase of iron in rat liver. Therefore, we hypothesized that intestinal iron concentration could be increased by copper-deficiency. Our objective was to examine the effects of iron as affected by dietary copper on beta-carotene 15,15'-dioxygenase activity in the small intestine. METHODS: Weanling male Sprague-Dawley rats (40 to 45g) were divided into four dietary groups: two copper-adequate groups (6.0 microg Cu/g diet) and two copper-deficient groups (0.6 microg Cu/g) combined with either normal iron (44 microg Fe/g) or high iron (87 microg Fe/g). Iron and copper concentrations were determined by atomic absorption spectrophotometry and the dioxygenase activity by reverse phase HPLC. RESULTS: Intestinal copper concentration was significantly reduced (40%) by the consumption of the copper-deficient diets, but intestinal iron was not changed by doubling dietary iron in rats fed either copper-adequate or copper-deficient diets. However, as hypothesized, the two copper-deficient groups exhibited higher intestinal iron concentration (> or =137%, p<0.001) than the copper-adequate controls. In addition, intestinal beta-carotene 15,15'-dioxygenase activity was increased by 27% and 106%, respectively, for copper-deficient rats fed either normal or high iron diets, compared to the respective copper-adequate controls (p<0.01). The dioxygenase activity was not significantly affected by dietary iron in either copper-adequate or copper-deficient groups. Finally, the enzyme activity was positively correlated (r=0.67, p<0.0001) with iron concentration and negatively correlated (r=-0.49, p<0.01) with copper concentration in small intestine. CONCLUSIONS: Intestinal beta-carotene 15,15'-dioxygenase may be an iron-dependent enzyme sensitive to copper status in vivo.     

Effects of inflammation and copper intake on rat liver and erythrocyte Cu-Zn superoxide dismutase activity levels

Stress such as inflammation produces an acute phase response that includes elevated levels of ceruloplasmin, the main copper component of plasma. Inflammatory effects on cellular copper enzyme activity levels are largely unknown. Cu-Zn superoxide dismutase (SOD) activities in liver, the main site of ceruloplasmin secretion, decreased with turpentine-induced inflammation (0.1 mL, intramuscular, leg) in rats fed any of three copper levels (adequate = 6 mg/kg, marginal = 2.5 mg/kg and deficient less than 0.5 mg/kg). Ceruloplasmin activities rose significantly with inflammation in the adequate and marginal groups but not in the deficient animals. Hepatic Cu-Zn SOD immunoreactive protein levels were unaffected by copper status or inflammatory state. Erythrocyte Cu-Zn SOD activities were influenced by dietary copper but not inflammation. An additional group of rats fed 15 mg copper/kg did not show a turpentine-induced decrease in liver Cu-Zn activity levels. Inflammatory effects on other copper enzyme activities did occur as evidenced by increases in ceruloplasmin and decreases in serum extracellular SOD. In conclusion, an acute phase response in rats increased the amount of dietary copper required to maintain hepatic Cu-Zn SOD activity at levels equal to those of nonstressed, copper-adequate rats. Rat erythrocyte Cu-Zn SOD activities provided a blood measurement reflective of copper intake with or without stress, but these values did not reflect decreases in liver Cu-Zn SOD activities after 3 d of inflammation.     

Use of immunoresponsiveness to demonstrate that the dietary requirement for copper in young rats is greater with dietary fructose than dietary starch

Weaned male Lewis rats were pair-fed diets containing 62.7% fructose or starch and either 6-7 mg Cu/kg diet (adequate) or 0.7 mg Cu/kg diet (deficient) for 33 d. Antibody titers after primary immunization with sheep erythrocytes were significantly lower in rats fed copper-deficient diets. Compared to starch, fructose markedly attenuated antibody production in copper-deficient rats. Dietary carbohydrate did not affect the humoral immune response of rats fed diets with adequate copper. Concentrations of copper in thymus, spleen, liver and heart were also significantly lower in rats fed fructose with deficient copper (F-Cu) than in the group fed starch with deficient copper. Thymic hypogenesis was observed only in the F-Cu group. Tissue concentrations of copper were reduced before antibody production was impaired. Repletion of previously copper-deficient rats rapidly restored immunocompetence and stimulated thymic growth. Immunoresponsiveness and tissue concentrations of copper reached control levels in rats fed diets containing starch and fructose at 1.2-1.6 and 2.2-2.9 mg Cu/kg diet, respectively. The results demonstrate that the amount of dietary copper required for optimal function of the humoral immune system, thymic growth and maintenance of normal tissue levels of this essential micronutrient is greater when young rats are fed diets with fructose than with starch.     

Comparison between dietary and cenetic copper deficiency in mice: Copper-dependent anemia

Copper deficiency was produced by feeding a low copper diet to mice and rats during gestation and lactation. The copper-deficient offspring were compared to Brindled mice, which exhibit copper-deficient characteristics due to a genetic defect within the X-chromosome. Weanling mice nursed by dams fed the low copper diet exhibit classical signs of copper deficiency previously reported in other species, including: anemia, hypoceruloplasminemia, depressed brain copper levels, and cytochrome oxidase activities. However, in comparison to weanling rats derived from dams given the same treatment, the mice show only a minimal growth deficit and marginal copper depletion of liver with normal cytochrome oxidase, activity. Younger 11-day-old copper-deficient male mice also develop anemia, have low tissue copper levels (liver and kidney) and exhibit decreased ceruloplasmin activity. However, unlike copper-deficient rats, these mice do not have a decrease in serum ferroxidase activity. Age-matched Brindled mice are not anemic, even though they have low serum ferroxidase activity. Similar to the dietary deficient mice, Brindled mice do have low ceruloplasmin activity and low liver copper levels. In an attempt to resolve this enigma, mouse serum was chromatographed on Sephadex G-150. Only one ferroxidase activity peak, clearly separated from ceruloplasmin, was observed. Human and rat sera display two ferroxidase activity peaks, one chromatographing with ceruloplasmin activity, which is sensitive to azide, and a second peak which, like the mouse serum peak, appears as a shoulder on the void volume. These results demonstrate the potential of this mouse model in elucidating the nature of copper-dependent anemia. Furthermore, the data from this model suggest that changes in serum ferroxidase activity cannot explain the existence of anemia in dietary copper deficiency nor absence of anemia in genetic copper deficiency (Brindled mice).     

Copper and iron deficiencies: effects on serum lipids and tissue minerals

To study the interrelationship between iron and copper on serum lipid concentrations, four diets were fed to growing rats: iron and copper deficient, copper-deficient, iron-deficient, iron and copper adequate. After 18 weeks, concentrations of iron and copper in organs and lipids in sera were determined. Iron deficiency alone or combined with copper deficiency resulted in reduced body weights, hemoglobin concentrations, hematocrits, and iron concentrations in liver, spleen, and heart. Hepatic copper was elevated 8-fold in iron deficiency. Copper deficiency alone or combined with iron deficiency resulted in reduced copper concentrations of liver and spleen and reduced ceruloplasmin. Serum triglycerides and cholesterol did not differ among experimental treatments. No significant effects of the interaction between dietary iron and copper on serum lipid levels were found.     

Influence of copper intake and inflammation on rat serum superoxide dismutase activity levels

Serum superoxide dismutase (SOD) activity concentrations, though small compared to tissue levels, could contribute to extracellular superoxide radical detoxification and act as indicators of copper status. The present study identified the response of rat serum SOD activity contents to marginal and deficient copper intakes and to inflammation. Rats fed copper-deficient diet (less than 0.2 mg/kg Cu) for 5 wk displayed serum SOD activity contents that were only about 20% of those found in rats fed copper-supplemented diet (6.0 mg/kg Cu). Activities in rats fed a marginal diet (1.5 mg/kg Cu) were about 55% of those in the adequate rats. Turpentine-induced inflammation lowered serum SOD in rats within each dietary group. However, the change in the marginal group was not statistically significant. Based on chromatographic characterizations and inhibitor studies, rat serum SOD activity seemed to result primarily from a copper protein other than ceruloplasmin, Cu-Zn SOD or the recently discovered tissue extracellular SOD. In conclusion, low copper intake and inflammation may compromise extracellular defenses against superoxide. In addition, serum SOD activities could provide a non-ceruloplasmin-related means of assessing copper status, but nondietary variables can also affect these SOD values.     

Effect of copper deficiency on erythrocyte membrane proteins of rats

The effects of copper deficiency on the proteins of rat erythrocyte membranes were assessed by electrophoretic analysis. For 42 d, rats were fed diets containing less than 1 ppm Cu with 35 or 250 ppm Fe or 5 ppm Cu with 35 or 250 ppm Fe. Electrophoresis of erythrocyte membrane proteins indicated a significant increase in the amount of a 170,000-dalton protein in rats fed copper-deficient diets. High dietary iron reduced the amount of the 170,000-dalton protein in erythrocyte membranes from rats in both the copper-deficient and copper-adequate groups. However, feeding high dietary iron to copper-deficient rats did not reduce the amount of the protein to the level found in rats fed the copper-adequate diet containing 35 ppm Fe. Triton X-100 extraction of erythrocyte membranes demonstrated that the 170,000-dalton protein was associated with the membrane cytoskeleton. Thus, copper deficiency possibly alters the cell's mechanical properties and consequently decreases erythrocyte survivability by modifying the membrane cytoskeleton.     

Induction of ceruloplasmin synthesis by retinoic acid in rats: influence of dietary copper and vitamin A status

Ceruloplasmin, a copper-containing acute phase plasma protein, has been shown to be regulated by 13-cis retinoic acid in rats. Ceruloplasmin activity was significantly increased within 24 h and remained elevated for at least 72 h after a single injection of 13-cis retinoic acid. With daily injections of retinoic acid, the ceruloplasmin activity continued to increase for at least 4 d. After 4 d, the activity was four times control levels. In copper-deficient rats, the ceruloplasmin activity did not increase in response to retinoic acid unless copper was also given to these rats 8 h after retinoic acid. Actinomycin D blocked the retinoic acid-induced stimulation of ceruloplasmin activity in copper-sufficient rats, but in copper-deficient rats only about half of the increase was blocked when the rats were given copper or copper and retinoic acid. By use of pulse-labeling techniques, ceruloplasmin synthesis was shown to increase 1.5-fold after retinoic acid and this increase was blocked by actinomycin D. When vitamin A-deficient rats were repleted with 13-cis retinoic acid for 3 or 5 d, both the ceruloplasmin activity and synthesis were significantly stimulated when compared to the nonrepleted, deficient rats. Therefore, the dietary components, copper and vitamin A, play an important role in the regulation of plasma ceruloplasmin levels.