Comparison of copper status in rats when dietary fructose is replaced by either cornstarch or glucose

The purpose of this study was to determine what levels of starch or glucose replacement for fructose in the copper-deficient diet (copper) can minimize the fructose-copper interaction. Experimental diets contained either 100% fructose as the carbohydrate source, or the fructose was partially replaced with 50% starch, 50% glucose, 75% starch, or 75% glucose. Diets were either copper adequate (7-8 ppm) or inadequate (less than 1 ppm). Male weanling rats were fed their respective diet for 5 weeks and then fasted overnight. After decapitation, blood was collected and liver and heart were removed. Plasma copper was significantly reduced and ceruloplasmin was not detected in all copper-deficient groups. Copper deficiency increased plasma cholesterol, as well as heart and liver weight in the glucose groups, but not in the starch groups. Those organ weights were heavier in glucose-copper than starch-copper rats. Erythrocyte copper-zinc-superoxide dismutase activity was greater in starch-copper rats. Erythrocyte copper-zinc-superoxide dismutase activity was greater in starch-copper than glucose-copper rats regardless of carbohydrate amount. Hepatic copper concentration of the group fed starch-copper was twice levels observed in glucose-copper. The 50% glucose rats had lower hepatic copper than the 75% glucose rats. Hepatic copper-zinc-superoxide dismutase activity showed patterns similar to hepatic copper. Cardiac copper was greater in starch-copper than glucose-copper rats. Cardiac copper-zinc-superoxide dismutase activity was equally reduced in all copper-deficient groups. The 50% starch-replaced diet was more effective in minimizing copper deficiency than the 75% glucose-replaced diet. This poorer improvement of copper deficiency by glucose than starch may partially be due to a more severe reduction of food intake in glucose than in starch diets.     

Interaction Between Dietary Carbohydrate and Copper Nutriture on Lipid Peroxidation in Rat Tissues

The effects of the interactions between dietary carbohydrates and copper deficiency on superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities and their roles in peroxidative pathways were investigated. Weanling rats were fed diets deficient in copper and containing either 62% starch, fructose, or glucose. Decreased activity of SOD was noted in all rats fed the copper-deficient diets regardless of the nature of dietary carbohydrate. However, the decreased activity was more pronouced in rats fed fructose. Feeding the fructose diets decreased the activity of GSH-Px by 25 and 50% in the copper-supplemented and copper-deficient rats, respectively, compared to enzyme activities in rats fed similar diets containing either starch or glucose. The decreased SOD and GSH-Px activities in rats fed the fructose diet deficient in copper were associated with increased tissue per-oxidation and decreased hepatic adenosine triphosphate (ATP). When the fructose in the diet of copper-deficient rats was replaced with either starch or glucose, tissue SOD and GSH-Px activities were increased and these increases in enzyme activity were associated with a tendency toward reduced mitochondrial peroxidation when compared to the corre-sponding values for rats fed fructose throughout the experiment Dietary fructose aggrevated the symptoms associated with copper deficiency, but starch or glucose ameliorated them. The protective effects were more pronounced with starch than with glucose.     

Fructose metabolizing enzymes in the rat liver and metabolic parameters: Interactions between dietary copper,type of carbohydrates,and gender

This study was conducted to determine the effects of nutrient interactions between dietary carbohydrates and copper levels on fructose-metabolizing hepatic enzymes in male and female rats. Male and female rats were fed diets for 5 weeks that were either adequate or deficient in copper that contained either starch or fructose. Rats of both sexes fed fructose as compared with those fed starch showed higher activity of hepatic fructose metabolizing enzymes. There were also significant differences in fructose metabolism of liver between the male and female rats. Female rats had lower hepatic ketohexokinase and triose kinase but higher triosephosphate isomerase activities compared with male rats. Male rats fed copper-deficient diets had lower aldolase B activity compared with those fed copper-adequate diets. Female rats fed copper-deficient diets had higher triosephosphate isomerase activity compared with rats fed copper-adequate diets. Our data suggest that gender differences in hepatic fructose metabolism may not be the primary reason for the severity of copper deficiency syndrome in male rats fed copper-deficient diet with fructose.     

Female rats are protected against the fructose induced mortality of copper deficiency

Experiments were conducted in copper deficient male and female rats fed diets containing fructose or starch in order to determine whether the same type of interaction between copper status and dietary carbohydrate found in male rats also occurs in the female rat. Mortality occurred only in the male rats fed the fructose diet deficient in copper with 40% of the animals dying during the 8 week study. Only anemia, hypercholesterolemia, increased BUN, heart hypertrophy and reduced body weight were observed in these animals which could be related to their mortality. Despite the increased mortality, plasma ceruloplasmin, erythrocyte SOD and hepatic copper concentrations were reduced to a similar extent in all rats regardless of the sex of the animals or of the type of dietary carbohydrate fed. The results of the present study indicate that although direct measurements of copper status of female rats fed fructose diet deficient in copper are similar to their male counterpart, they are apparently protected from the lethal consequences of the deficiency.     

Effect of dietary carbohydrates and copper status on blood pressure of rats

Copper deficiency was induced in rats by feeding diets containing either 62% starch, fructose or glucose deficient in copper for 6 weeks. All copper deficient rats, regardless of the dietary carbohydrate, exhibited decreased ceruloplasmin activity and decreased serum copper concentrations. Rats fed the fructose diet exhibited a more severe copper deficiency as compared to rats fed either starch or glucose. The increased severity of the deficiency was characterized by reduced body weight, serum copper concentration and hematocrit. In all rats fed the copper adequate diets, blood pressure was unaffected by the type of dietary carbohydrate. Significantly reduced systolic blood pressure was evident only in rats fed the fructose diet deficient in copper. When comparing the three carbohydrate diets, the physiological and biochemical lesions induced by copper deprivation could be magnified by feeding fructose.     

Effects of dietary tin and copper on rat hepatocellular antioxidant protection

The effects of dietary tin on copper status and on enzymes and metabolites involved in hepatocellular antioxidant protection were measured in rats fed copper-adequate or copper-deficient diets with glucose or fructose. Rats became copper-depleted after 4 weeks on diets containing less than 0.5 micrograms of copper/g as evidenced by significant decreases in liver copper and serum ceruloplasmin. Signs of copper deficiency occurred in copper-depleted rats fed diets containing 100 micrograms of tin/g. Significant effects of tin on liver glutathione peroxidase and superoxide dismutase activities and on liver iron and total glutathione concentrations were observed. Interactions between copper and tin on liver copper and iron and on liver superoxide dismutase and malondialdehyde production are reported. Adverse effects of feeding diets containing 100 micrograms of tin/g include (i) copper depletion in rats fed copper-adequate diets, (ii) accelerated development of copper deficiency in rats fed copper-deficient diets, and (iii) reduction in hepatocellular antioxidant protection.     

Effects of different dietary carbohydrates on hepatic enzymes of copper-deficient rats

The present study was undertaken to measure the activities of several hepatic enzymes of regulatory importance in the pathways of lipogenesis and gluconeogenesis in rats fed diets marginally deficient in copper (1.2 micrograms Cu/g of diet) and containing either fructose, glucose, or starch as the carbohydrate sources. Although all copper-deficient rats exhibited the characteristic signs of copper deficiency, they were more pronounced in rats fed the diet containing fructose. Except for the activity of phosphoenolpyruvate carboxykinase which was unaffected either by copper deficiency or by the type of dietary carbohydrate, the hepatic activities of glucose-6-phosphate dehydrogenase, malic enzyme, L-alpha-glycerophosphate dehydrogenase and fructose 1,6-diphosphatase were unaffected by copper deficiency but were affected by the type of carbohydrate in the diet. Fructose produced the greatest increase in enzymatic activities, whereas starch produced the least activity and glucose induced an intermediate effect. These results indicate that the deleterious effects of a fructose diet deficient in copper on biochemical and physiological indices could not be due to an immediate metabolite of fructose. However, the involvement of a subsequent metabolite of fructose in the mechanism of copper utilization and/or requirement cannot be excluded.     

Effect of nutritional copper deficiency on carrageenin edema in the rat

The effects of nutritional copper deficiency on carrageenin edema in the rat were investigated with emphasis on studying the correlation between the degree of copper deficiency and the degree of edema. Carrageenin paw edema in both copper-sufficient and copper-deficient groups of rats was compared after either 20, 40, or 60 d on respective diets. The degree of copper deficiency was quantitated by analyzing total copper concentrations in a number of tissues. Other copper dependent parameters were also determined. Results indicated that: (1) although copper sufficient rats showed relatively little change in the degree of edema, copper-deficient rats showed a steady and significant increase in edema from d 20 to 40 to 60; (2) paw edema in copper-deficient animals was highly and negatively correlated to the concentrations of copper in the liver; the correlation with liver Cu,Zn-superoxide dismutase activity, however, was inconsistent; (3) paw edema was not correlated either to copper concentration in tissues other than liver or to plasma ceruloplasmin activity; and (4) aggravation of carrageenin edema in copper-deficient animals seemed to be mediated via an as yet unknown secondary effect of copper deficiency.     

Dietary carbohydrate influences tissue fatty acid and lipid composition in the copper-deficient rat

Fructose and copper have been shown independently to influence long chain fatty acid metabolism. Since fructose feeding exacerbates copper deficiency, their possible interaction with respect to tissue long chain fatty acid and lipid composition was studied. Weanling male Sprague-Dawley rats were given diets containing 0.6 or 6 mg/kg copper. The carbohydrate source (627 g/kg) was either fructose or corn starch. After 3 wk, fatty acid profiles and total lipids in heart and liver were analyzed. Copper-deficient rats fed fructose had more severe signs of copper deficiency than those fed starch, according to heart/body wt ratio, hematocrit, and liver copper content. The fatty acid composition of heart and liver triacylglycerol was significantly different between groups, but the changes did not correlate with the severity of copper deficiency. In heart, phosphatidylinositol and phosphatidylserine, arachidonic acid and docosapentaenoic acid (n-6) were increased 193 and 217%, respectively, p<0.05) in rats given the copper-deficient diet containing fructose. Changes in the long chain fatty acids in heart phospholipids may be related to the higher mortality commonly observed in rats fed a copper-deficient diet containing fructose.     

The effect of copper deficiency on the formation of hemosiderin in sprague-dawley rats

We demonstrated previously that loading iron into ferritin via its own ferroxidase activity resulted in damage to the ferritin while ferritin loaded by ceruloplasmin, a copper-containing ferroxidase, was not damaged and had similar characteristics to native ferritin (Welch et al. (2001) Free Radic Biol Med 31:999–1006). Interestingly, it has been suggested that the formation of hemosiderin, a proposed degradation product of ferritin, is increased in animals deficient in copper. In this study, groups of rats were fed normal diets, copper deficient diets, iron supplemented diets, or copper deficient-iron supplemented diets for 60 days. Rats fed copper-deficient diets had no detectable active serum ceruloplasmin, which indicates that they were functionally copper deficient. There was a significant increase in the amount of iron in isolated hemosiderin fractions from the livers of copper-deficient rats, even more than that found in rats fed only an iron-supplemented diet. Histological analysis showed that copper-deficient rats had iron deposits (which are indicative of hemosiderin) in their hepatocytes and Kupffer cells, whereas rats fed diets sufficient in copper only had iron deposits in their Kupffer cells. Histologic evidence of iron deposition was more pronounced in rats fed diets that were deficient in copper. Additionally, sucrose density-gradient sedimentation profiles of ferritin loaded with iron in vitro via its own ferroxidase activity was found to have similarities to that of the sedimentation profile of the hemosiderin fraction from rat livers. The implications of these data for the possible mechanism of hemosiderin formation are discussed.     

Renal copper as an index of copper status in marginal deficiency

Marginal copper (Cu) deficiency is difficult to study, in part because its effects may be small, but also because feeding of a deficient diet may not cause a discernable change in Cu status. The key to resolution of effects may be in the choice of Cu status index. In this study, liver Cu concentration, a commonly used index of Cu status, was compared with activity of ceruloplasmin (CP), a circulating Cu-dependent enzyme, and kidney Cu concentration for their utility in resolving effects of marginal Cu deficiency. Seventy male, weanling rats were fed diets containing, nominally, 0, 1.5, 3, 4.5, or 6 mg Cu/kg diet for 5 wk. All three indices showed strong depression with severe deficiency (dietary Cu=0), but were relatively weak in their ability to distinguish between animals fed marginally deficient diets when compared by group statistics (ANOVA). Further, group statistics revealed no effect of marginal deficiency on six other variables known to change with severe Cu deficiency: heart weight/body weight, hematocrit, red cell distribution width, neutrophil count, glycated hemoglobin, and platelet count. To take into account interanimal variation, the three putative indices were plotted against these six variables and linear regression was performed on points representing marginally deficient rats. None of the variables showed significant regression with liver Cu or serum ceruloplasmin, but three showed significant regression with kidney Cu. These findings indicate that kidney Cu is preferable to liver Cu or ceruloplasmin as an index of Cu status in marginal deficiency and that linear regression is a possible way of testing for effects of marginal Cu deficiency, especially when effects are subtle.     

Sexual differences in the expression of copper deficiency in rats

The present investigation was undertaken to establish whether the severity of copper deficiency in rats fed diets containing fructose is affected by the presence and type of endogenous sex hormones. Intact and castrated male rats and intact and ovariectomized females were fed from weaning a copper-deficient diet (0.6 ppm) containing 62% fructose for 8 weeks. Regardless of castration, male rats were anemic, exhibited heart hypertrophy, and died of the deficiency. However, castration ameliorated the anemia and delayed the mortality. In contrast, none of the females died of the deficiency. It is suggested that in addition to the sex of the animal, levels of testosterone in the male may also play a role in the severity of copper deficiency.     

Dietary copper, simple sugars, and metabolic changes in pigs

Inadequate dietary copper is known to result in undesirable metabolic changes in rats and humans. Abnormal cardiac function, leading to sudden death, is a common finding when copper deficient rats are fed a 62% fructose diet. To further study the apparent mineral-carbohydrate relationship to cardiac physiology, 3 male and 3 female swine were randomly assigned to four groups (6 pigs per group) which were fed low copper (1.5 ppm) or copper supplemented (40 ppm) diets with 20% of calories from either fructose or glucose for 10 weeks. In agreement with results from other animal studies, copper deficient swine exhibited decreased plasma ceruloplasmin, erythrocyte superoxide dismutase and plasma lysyl oxidase activities and lowered serum copper. The copper deficient fructose group had the lowest aortic lysyl oxidase activity and hematocrit when compared to the other groups. The relative heart weight in the copper deficient fructose group was 93% greater than the other three dietary groups. The livers of copper deficient fructose fed pigs were also significantly larger. Two enzymes related to cardiac and hepatic function, aspartate and alanine aminotransferase were also measured. Copper deficiency significantly lowered alanine aminotransferase but there was no dietary effect on aspartate amino-transferase. The results of this project indicate that the pig is a sensitive model for the study of cardiovascular abnormalities which occur when fructose is consumed with a low copper diet.     

Silicon facilitation of copper utilization in the rat

An eight-week, 2 x 4 factorial rat experiment using two levels of dietary copper and four levels of dietary silicon was conducted to further delineate a previously observed silicon-copper interaction in which silicon appears to mimic copper in its effect on the composition of the aorta. Dietary copper concentrations were 1.4 (deficient) and 5.4 (adequate) mg/kg diet, and silicon concentrations were 5, 135, 270, and 540 mg/kg diet. Compared with the lowest level of silicon and copper, weight gains were 15.5% higher for rats fed 540 mg silicon/kg diet and 14.3% higher for those fed 5.4 mg copper/kg diet. The growth-promoting effects of silicon and copper were additive. Evidence that silicon elevated the copper status of copper-deficient rats includes an increase in packed-cell volume by 540 mg silicon/kg diet in the otherwise packed-cell volume-depressed, copper-deficient rats, accompanied by a trend toward higher hemoglobin values and lower relative heart weights. In the copper-adequate rats, evidence that 540 mg silicon/kg diet elevated their copper status includes a two-fold increase in the blood-plasma copper concentration, a three-fold increase in ceruloplasmin activity, and an increase in cardiac, renal, and hepatic copper concentrations. In addition, 540 mg silicon/kg diet resulted in higher aortic dry mass and aortic elastin content in both copper-deficient and copper-adequate rats. While dietary silicon concentrations of 135, 270, and 540 mg/kg diet were all effective in increasing aortic elastin in the copper-adequate rats, only 540 mg silicon/kg diet increased aortic elastin in the copper-deficient rats. These data indicate that some of the metabolic effects attributed to silicon may be manifested through a silicon-facilitated increase in copper utilization.     

Nutritional copper deficiency does not affect sponge granuloma formation in the rat

Sponge granuloma formation was compared in copper-deficient and copper-sufficient rats following feeding of respective diets for 20, 40, or 60 d. Body weight, total blood hemoglobin, and activities of ceruloplasmin and Cu, Zn-superoxide dismutase in plasma were monitored to ascertain copper deficiency. Mean granuloma weights (mg +/- SEM) in copper-deficient and copper-sufficient groups of rats, respectively, were as follows: 37 +/- 2 and 38 +/- 2 after 20 d, 22 +/- 2 and 23 +/- 2 after 40 d, and 19 +/- 1 and 21 +/- 1 after 60 d on respective diets. Thus, nutritional copper deficiency did not have an effect on sponge granuloma formation in the rat.     

Folate and homocysteine metabolism in copper-deficient rats.

To investigate the effect of copper deficiency on folate and homocysteine metabolism, we measured plasma, red-cell and hepatic folate, plasma homocysteine and vitamin B-12 concentrations, and hepatic methionine synthase activities in rats. Two groups of male Sprague-Dawley rats were fed semi-purified diets containing either 0. 1 mg (copper-deficient group) or 9.2 mg (control group) of copper per kg. After 6 weeks of dietary treatment, copper deficiency was established as evidenced by markedly decreased plasma and hepatic copper concentrations in rats fed the low-copper diet. Plasma, red-cell, hepatic folate, and plasma vitamin B-12 concentrations were similar in both groups, whereas plasma homocysteine concentrations in the copper-deficient group were significantly higher than in the control group (P<0.05). Copper deficiency resulted in a 21% reduction in hepatic methionine synthase activity as compared to the control group (P<0.01). This change most likely caused the increased hepatic 5-methyltetrahydrofolate and plasma homocysteine concentrations in the copper-deficient group. Our results indicate that hepatic methionine synthase may be a cuproenzyme, and plasma homocysteine concentrations are influenced by copper nutriture in rats. These data support the concept that copper deficiency can be a risk factor for cardiovascular disease.     

Consequences of copper deficiency are not differentially influenced by carbohydrate source in young pigs fed a dired skim milk-based diet

Carbohydrates (CHO) such as fructose (FR) or sucrose, but not starch (ST), aggravate the consequences of dietary copper (Cu) deficiency in rats. To evaluate whether this Cu X CHO interaction is pertinent to human health, the pig was used as an animal model. In two studies, 66 weanling pigs were fed dried skim milk (DSM)-based diets for 10 wk with 20% of the total calories provided as either FR, glucose, or ST and containing either deficient (1.0-1.3 micrograms/g diet) or adequate (7.1 micrograms/g) levels of Cu. Plasma and tissue levels of Cu, the activities of plasma ceruloplasmin ferroxidase and erythrocyte Cu, Zn-superoxide dismutase, and hematocrits were lower (p less than 0.05) in animals fed Cu-deficient diets. The relative cardiac mass of all Cu-deficient groups was greater (p less than 0.05) than that of animals fed Cu-adequate diets. These effects were in general unaffected by type of CHO. For comparison, weaned male rats were also fed DSM-based containing diets ST or FR with adequate or deficient Cu for as long as 10 wk. Rats consuming the Cu-deficient diets were characterized by significantly lower hematocrits, decreased tissue Cu levels, and enlarged hearts, regardless of the CHO source. Together, these data demonstrate that DSM-based diets are not suitable for delineation of potential Cu X CHO interactions, and one or more components of DSM may exacerbate the consequences of dietary Cu deficiency.     

Inhibition of elevated hepatic glutathione abolishes copper deficiency cholesterolemia.

Dietary copper deficiency causes hypercholesterolemia and increased hepatic 3-hydroxy-3-methyl-glutaryl coenzyme A (MHG-CoA) reductase activity and increased hepatic glutathione (GSH) in rats. We hypothesized that inhibition of GSH production by L-buthionine sulfoximine (BSO), a specific GSH synthesis inhibitor, would abolish the cholesterolemia and increased HMG-CoA reductase activity of copper deficiency. In two experiments, two groups of 20 weanling male rats were fed diets providing 0.4 and 5.8 micrograms Cu/g, copper-deficient (Cu-D) and copper-adequate (Cu-A), respectively. At 35 days plasma cholesterol was significantly elevated by 30 to 43% in Cu-D and 10 animals in each of the Cu-D and Cu-A groups were randomly assigned to receive 10 mM BSO solution in place of drinking water and continued on the same diets for another 2 wk. At necropsy Cu-D animals had a significant 52 to 58% increase in plasma cholesterol. BSO administration abolished the cholesterolemia in Cu-D rats, but had no influence on plasma cholesterol of Cu-A rats. Hepatic GSH was increased 39 to 82% in Cu-D rats and BSO abolished this increase. BSO was without effect on cardiac hypertrophy, plasma and liver copper, and hematocrit indices of copper status. Liver microsome HMG-CoA reductase activity was significantly increased 85 to 288% in Cu-D rats and BSO administration abolished this increase in activity in Cu-D rats. The results suggest that copper deficiency cholesterolemia and elevated HMG-CoA reductase activity are a consequence of elevated hepatic GSH, and provide evidence for GSH regulation of cholesterol metabolism in intact animals.     

The effect of vitamin E on lipid peroxidation in the copper-deficient rat

The present study was designed to determine whether the supplementation of vitamin E in the copper-deficient diet would ameliorate the severity of copper deficiency in fructose-fed rats. Lipid peroxidation was measured in the livers and hearts of rats fed a copper-deficient diet (0.6 microg Cu/g) containing 62% fructose with adequate vitamin E (0.1 g/kg diet) or supplemented with vitamin E (1.0 g/kg diet). Hepatic lipid peroxidation was significantly reduced by vitamin E supplementation compared with the unsupplemented adequate rats. In contrast, myocardial lipid peroxidation was unaffected by the level of vitamin E. Regardless of vitamin E supplementation, all copper-deficient rats exhibited severe signs of copper deficiency, and some of the vitamin E-supplemented rats died of this deficiency. These findings suggest that although vitamin E provided protection against peroxidation in the liver, it did not protect the animals against the severity of copper deficiency induced by fructose consumption.     

The effect of copper deficiency on rat hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase activity

The effect of copper deficiency on hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase, the key enzyme regulating cholesterol biosynthesis, was investigated in the rat. Male weanling rats were fed semipurified diets containing adequate, marginal, or deficient levels of copper for 6 weeks. Two separate studies were conducted; in the first study, animals were fasted 12 hours prior to analysis and in the second study, animals were fed diets ad libitum. Plasma lipid levels, hepatic cholesterol concentrations, and 3-hydroxy-3-methylglutaryl coenzyme A reductase specific activity, total and active, were determined. Consistent with previous findings, plasma total cholesterol and triglyceride levels were significantly elevated in copper-deficient rats. Copper deficiency resulted in a significant decrease in hepatic total cholesterol levels. Total and active levels of 3-hydroxy-3-methylglutaryl coenzyme A reductase in fed animals were elevated twofold with copper deficiency, with the active form of the enzyme constituting approximately 30% of total activity. 3-Hydroxy-3-methylglutaryl coenzyme A reductase activity in copper-deficient fasted rats was twofold higher than for the fasted adequate animal; however, fasting did result in a 10-fold reduction in hepatic reductase specific activity. These data support the hypothesis that copper deficiency results in a hypercholesterolemic state in the rat associated with increased hepatic cholesterol synthesis.