Interactive effects of dietary silicon, copper, and zinc in the rat

A factorial rat experiment using two dietary concentrations each of copper, zinc, and silicon was conducted to identify areas in which interrelationships involving silicon may exist. The concentrations used were (mg/kg of diet): copper, 1 and 5; zinc, 2 and 12; and silicon, 5 and 270. An antagonism between silicon and zinc, whereby increases in dietary levels of either one resulted in a reduction in blood plasma concentrations of the other, was demonstrated. The depressing effect of silicon on plasma concentrations of zinc and on alkaline phosphatase occurred only in zinc-deficient rats. However, silicon had no effect on growth. Effects on aortic composition, interpreted as beneficial, accompanied increases in the silicon content of copper-deficient diets. Silicon-dependent increases in the chloroform-methanol extractable fraction of aorta closely approximated a similar response to copper. High dietary silicon increased aortic elastin in copper-deficient rats when dietary zinc was adequate. The aortic effects of silicon, while mimicking the gross effects of copper, occurred in the absence of any silicon-related changes in blood copper concentrations. Interrelationships of silicon with other elements, particularly copper and zinc, may warrant consideration in future nutritional and metabolic studies.     

Early and advanced glycation end-products are increased in dietary copper deficiency

The hypothesis that nonenzymatic glycosylation of proteins (glycation) contributes to damage associated with dietary copper deficiency has depended largely on indirect evidence. Thus far, the observation of an elevated percentage of glycated hemoglobin in copper-deficient rats has provided the only direct evidence of an increase in glycation. We sought further direct evidence of increased glycation in copper deficiency. Male weanling rats were fed a copper-adequate (CuA, 6.4 mg Cu/kg diet) or copper-deficient diet (CuD, 0.4 mg Cu/kg diet) for 5 weeks. Rats fed the CuD diet were copper deficient as judged by depressed organ copper concentrations and a variety of indirect indices. Measurements of hemoglobin A(1) and serum fructosamine (both early glycation end-products) as well as serum pentosidine (an advanced glycation end-product) indicated that all three compounds were elevated in CuD rats relative to CuA rats. This finding further supports the view that glycation is enhanced and thus may contribute to defects associated with dietary copper deficiency.     

In vivo apoprotein catabolism of high density lipoproteins in copper-deficient, hypercholesterolemic rats

High density lipoprotein (HDL) apoprotein catabolism was examined in male Sprague-Dawley rats deficient in dietary copper. Twenty-four rats were randomly divided into two groups: copper-adequate (control, 5 mg of copper/kg diet) and copper-deficient (0.6 mg of copper/kg diet). After 5 weeks, animals were administered a tracer dose of iodinated HDL protein previously isolated from donor rats that were subjected to the same dietary treatments as the test animals. Copper-deficient rats exhibited a 54% increase in plasma volume and a 26% increase in HDL protein concentration above controls. Consequently, the intravascular pool of total HDL protein was increased 2-fold. The fractional catabolic rate of total HDL protein was similar between groups. However, because of the increased intravascular HDL pool in copper-deficient animals, the absolute catabolic rate was greater (640 +/- 49 micrograms/hr vs 316 +/- 12 micrograms/hr in controls). Tissue uptake of total HDL protein in copper-deficient rats tended to be greater in the kidneys, spleen, and testes compared with controls; the heart exhibited a significant 2.3-fold increase. In contrast, the catabolic rate of HDL protein in the liver and adrenal gland were not different between treatment groups. That an obligatory increase in HDL protein uptake was not observed in the liver and adrenal gland (organs which are sensitive to and can further metabolize cholesterol) suggests that these organs may be regulated, possibly contributing to the observed hypercholesterolemia in this model. These data imply that total HDL apoprotein catabolism is increased in response to the increased intravascular pool of HDL in copper-deficient rats.     

The Cardiac Copper Chaperone Proteins Sco1 and CCS are Up-Regulated,but Cox 1 and Cox4 are Down-Regulated,by Copper Deficiency

Copper is ferried in a cell complexed to chaperone proteins, and in the heart much copper is required for cytochrome c oxidase (Cox). It is not completely understood how copper status affects the levels of these proteins. Here we determined if dietary copper deficiency could up- or down-regulate select copper chaperone proteins and Cox subunits 1 and 4 in cardiac tissue of rats. Sixteen weanling male Long–Evans rats were randomized into treatment groups, one group receiving a copper-deficient diet (<1 mg Cu/kg diet) and one group receiving a diet containing adequate copper (6 mg Cu/kg diet) for 5 weeks. Hearts were removed, weighed, and non-myofibrillar proteins separated to analyze for levels of CCS, Sco1, Ctr1, Cox17, Cox1, and Cox4 by SDS–PAGE and Western blotting. No changes were observed in the concentrations of CTR1 and Cox17 between copper-adequate and copper-deficient rats. CCS and Sco1 were up-regulated and Cox1 and Cox4 were both down-regulated as a result of copper deficiency. These data suggest that select chaperone proteins and may be up-regulated, and Cox1 and 4 down-regulated, by a dietary copper deficiency, whereas others appear not to be affected by copper status.     

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.     

Interaction between nickel and copper in the rat

Two 42-d experiments were conducted with weanling male rats to study interactions between nickel and copper. In Experiment 1, a low-copper basal diet was supplemented with copper at 0 or 30 ppm and nickel at 0 or 30 ppm. Copper was added in Experiment 2 to a basal copper-deficient diet at a level of 0 or 15 ppm and nickel was supplemented at 0, 15, or 225 ppm. Responses to dietary nickel were dependent upon copper nutriture and experimental duration. Nickel had little effect on growth during the first 21 d of either study when added at low levels (15 or 30 ppm) to copper-deficient diets. Nickel supplementation depressed gains between 21 and 42 d in rats fed copper-deficient, but not copper-adequate, diets. Hematocrits and hemoglobin concentrations were not significantly affected by dietary nickel at 21 d. Nickel supplementation decreased hematocrits and hemoglobin values in copper deficient rats at 42 d in Experiment 1, but not in Experiment 2. Absorption of copper apparently was not reduced by nickel, since tissue copper concentrations were generally not decreased by increasing dietary nickel. Nickel supplementation increased lung and heart copper concentrations in Experiment 2. Liver iron was not affected by nickel, but spleen iron concentrations were reduced by nickel supplementation in copper-deficient rats in Experiment 2. The present studies suggest that nickel acts antagonistically to copper in certain biological processes.     

Marginal copper deficiency and atherosclerosis

Copper is an essential trace element in the maintenance of the cardiovascular system. Copper-deficient diets can elicit, in animals, structural and functional changes that are comparable to those observed in coronary heart disease. In this study, the effect of dietary-induced copper deficiency on aortic lesion development was measured by quantitative image analysis in C57BL/6 mice that are susceptible to diet-induced aortic lesions. The diets administered were severely copper deficient (0.2 mg/kg diet), marginally deficient (0.6 mg/kg diet), or copper adequate (6.0 mg/kg diet). Similarly, increased aortic lesion areas and elevated serum cholesterol were demonstrated in both deficient groups, compared with the copper-adequate group. Evidence for graded differences in copper status among the dietary groups was shown by the dose-response increase in liver copper concentration, copper-zinc superoxide dismutase and cytochrome-c oxidase activities, together with serum caeruloplasmin oxidase with increasing intakes of dietary copper. Despite the difference in copper status between the copper marginal and severely deficient groups, similar lesions found in both groups of mice suggest a threshold effect of copper deficiency on lesion formation.     

Biochemical interactions among silicon, iron and ascorbic acid in the rat

A 2 x 2 x 2 factorial experiment was conducted using two dietary levels each (mg/kg of diet) of silicon, 0 and 500; iron, 35 and 187; and ascorbic acid, 0 and 900, to identify biochemical interactions occurring among these nutrients. Supplemental silicon, in conjunction with the higher dietary-iron level, prevented the plasma-iron decreasing effect observed for the higher level of iron in the absence of silicon. In the absence of ascorbic acid, silicon also increased iron concentration in the liver. Lower growth of the silicon and iron-supplemented rats is believed to be a response to a subsequent iron-imposed aberration of copper or zinc metabolism. This is supported by decreased intestinal metallothionein, increased weights (g/100 g body weight) of liver, heart, and testes, and decreased packed-cell volume and hemoglobin concentration. The lower plasma-iron level associated with the higher level of dietary iron appeared to be an expression of the iron-imposed reduction of liver copper stores. Ascorbic acid decreased plasma-iron concentration and prevented the silicon-related increase in liver iron.     

Cu,Zn-superoxide dismutase is lower and copper chaperone CCS is higher in erythrocytes of copper-deficient rats and mice

Discovery of a sensitive blood biochemical marker of copper status would be valuable for assessing marginal copper intakes. Rodent models were used to investigate whether erythrocyte concentrations of copper,zinc-superoxide dismutase (SOD), and the copper metallochaperone for SOD (CCS) were sensitive to dietary copper changes. Several models of copper deficiency were studied in postweanling male Holtzman rats, male Swiss Webster mice offspring, and both rat and mouse dams. Treatment resulted in variable but significantly altered copper status as evaluated by the presence of anemia, and lower liver copper and higher liver iron concentrations in copper-deficient compared with copper-adequate animals. Associated with this copper deficiency were consistent reductions in immunoreactive SOD and robust enhancements in CCS. In most cases, the ratio of CCS:SOD was several-fold higher in red blood cell extracts from copper-deficient compared with copper-adequate rodents. Determination of red cell CCS:SOD may be useful for assessing copper status of humans.     

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.     

Tropoelastin production and tropoelastin messenger RNA activity. Relationship to copper and elastin cross-linking in chick aorta.

The elastin content of the chick thoracic aorta increases 2--3-fold during the first 3 weeks post-hatching. The deposition of elastin requires the covalent cross-linking of tropoelastin by means of lysine-derived cross-links. This process is sensitive to dietary copper intake, since copper serves as cofactor for lysyl oxidase, the enzyme that catalyses the oxidative deamination of the lysine residues involved in cross-link formation. Disruption of cross-linking alters tissue concentrations of both elastin and tropoelastin and results in a net decrease in aortic elastin content. Autoregulation of tropoelastin synthesis by changes in the pool sizes of elastin or tropoelastin has been suggested as a possible mechanism for the diminished aortic elastin content. Consequently, dietary copper deficiency was induced to study the effect of impaired elastin cross-link formation on tropoelastin synthesis. Elastin in aortae from copper-deficient chicks was only two-thirds to one-half the amount measured in copper-supplemented chicks, whereas copper-deficient concentrations of tropoelastin in aorta were at least 5-fold higher than normal. In spite of these changes, however, increased amounts of tropoelastin, copper deficiency and decreased amounts of elastin did not influence the amounts of functional elastin mRNA in aorta. Likewise, the production of tropoelastin in aorta explants was the same whether the explants were taken from copper-sufficient or -deficient birds. The lower accumulation of elastin in aorta from copper-deficient chicks appeared to be due to extracellular proteolysis, rather than to a decrease in the rate of synthesis. Electrophoresis of aorta extracts, followed by immunological detection of tropoelastin-derived products, indicated degradation products in aortae from copper-deficient birds. In extracts of aortae from copper-sufficient chicks, tropoelastin was not degraded and appeared to be incorporated into elastin without further proteolytic processing.     

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.     

Copper activation of superoxide dismutase in rat erythrocytes

Several lines of evidence suggested that copper can activate a preexisting pool of superoxide dismutase (SOD) apoprotein in erythrocytes from copper-deficient rats. First, feeding adequate copper to copper-deficient rats raised initially low erythrocyte SOD activities to normal values in under one-third the time needed to replace the entire red cell population. Moreover, copper injection (1 mg Cu/kg, sc) doubled erythrocyte SOD activity levels in 16 h. Since protein synthesis is restricted in mature erythrocytes, these results imply that copper activated apoSOD in vivo. Furthermore, injected copper raised SOD activity contents of both young and old erythrocytes. Neither dietary copper status nor copper injection influenced red cell SOD immunoreactive protein levels. In contrast, copper injection increased the amount of copper associated with the SOD activity peak region resulting from gel filtration of hemoglobin-free erythrocyte proteins on Sephadex G-75. Copper ions (3 microM) elevated SOD activity levels in vitro by 63% in 4 h in intact red cells from copper-deficient rats. No activation took place in lysed red cells from the same rats or in intact cells from copper-adequate rats. These results all suggest that copper can activate SOD apoprotein in erythrocytes by a specific, saturable process.     

Decreased passive stiffness of cardiac myocytes and cardiac tissue from copper-deficient rat hearts

Passive stiffness characteristics of isolated cardiac myocytes, papillary muscles, and aortic strips from male Holtzman rats fed a copper-deficient diet for approximately 5 wk were compared with those of rats fed a copper-adequate diet to determine whether alterations in these characteristics might accompany the well-documented cardiac hypertrophy and high incidence of ventricular rupture characteristic of copper deficiency. Stiffness of isolated cardiac myocytes was assessed from measurements of cellular dimensional changes to varied osmotic conditions. Stiffness of papillary muscles and aortic strips was determined from resting length-tension analyses and included steady-state characteristics, dynamic viscoelastic stiffness properties, and maximum tensile strength. The primary findings were that copper deficiency resulted in cardiac hypertrophy with increased cardiac myocyte size and fragility, decreased cardiac myocyte stiffness, and decreased papillary muscle passive stiffness, dynamic stiffness, and tensile strength and no alteration in aortic connective tissue passive stiffness or tensile strength. These findings suggest that a reduction of cardiac myocyte stiffness and increased cellular fragility could contribute to the reduced overall cardiac tissue stiffness and the high incidence of ventricular aneurysm observed in copper-deficient rats.     

Dietary copper and dimethylhydrazine affect protein kinase C isozyme protein and mRNA expression and the formation of aberrant crypts in colon of rats.

Low dietary copper has been shown to decrease the expression of various protein kinase C (PKC) isozymes and increase the risk of colon cancer development in experimental animals. The purpose of this study was to investigate the relationship between dietary copper and carcinogen administration on PKC isozyme accumulation and aberrant crypt foci (ACF) formation in rats fed 0.9 and 7.7 microg Cu/g diet. After 24 and 31 d on the diets, the rats were injected with either dimethylhydrazine (DMH) (25 mg/kg i.p.) or saline and killed at two time points (2 wk and 8 wk after DMH). Rats fed low dietary copper had significantly lower (p<0.0001) hematocrits, hemoglobin, ceruloplasmin activity and plasma and liver copper concentrations than rats fed adequate dietary copper. Ingestion of low dietary copper significantly (p<0.005) increased the formation of DMH-induced ACF (116.8 vs 59.6). Low dietary copper significantly (p<0.05) decreased the concentration of PKC alpha, delta, and zeta in the colon at 2 wk but not at 8 wk. Thus, changes in PKC isoform protein concentration may be related to increased susceptibility of copper-deficient animals to colon cancer.     

Regional Specificity in Alterations of Rat Brain Copper and Catecholamines Following Perinatal Copper Deficiency

Abstract: Perinatal copper deficiency was studied in 1-month-old female and male Sprague-Dawley rat offspring to investigate regional changes in brain copper and catecholamine levels. Offspring of dams given the low copper treatment beginning at day 7 of gestation exhibited signs characteristic of deficiency such as impaired growth and 10-fold lower liver copper levels compared with copper-adequate controls. Regional analysis of brain copper by graphite furnace atomic absorption spectroscopy revealed uniform and severe reduction of copper to levels 20 ± 3% of controls in all regions, except the hypothalamus, where reductions to 56 and 28% of those in copper-adequate females and males, respectively, were measured. HPLC analysis revealed significant reductions in norepinephrine levels in cerebrum, midbrain, corpus striatum, cerebellum, and medulla-pons of copper-deficient offspring ranging between 39 and 67% of control values. There were no significant differences in norepinephrine concentration in the hypothalamus. There was a significant, one-third reduction of dopamine in the corpus striatum of copper-deficient male rats. Consistent with altered in vivo dopamine β-monooxygenase activity, there were five-, three-, and twofold elevations of dopamine in cerebellum, medulla-pons, and hypothalamus of copper-deficient rats. Spectrophotometric measurement of in vitro dopamine β-monooxygenase activity of brain and adrenal homogenates was higher in copper-deficient rats, confirming prior work. An explanation for the in vitro data is unclear. Changes in copper and catecholamine levels were influenced by diet and were regionally selective, especially in the hypothalamus.     

Impaired cardiac mitochondrial membrane potential and respiration in copper-deficient rats

Cardiac mitochondrial respiration, ATP synthase activity, and membrane potential and intactness were evaluated in copper-deficient rats. In the presence of NADH, both copper-deficient and copper-adequate mitochondria had very low oxygen consumption rates, indicating membrane intactness. However copper-deficient mitochondria had significantly lower oxygen consumption rates with NADH than did copper-adequate mitochondria. Copper-deficient mitochondria had significantly lower membrane potential than did copper-adequate mitochondria using fluorescent dyes. Copper-deficient mitochondria had significantly lower state 3 oxygen consumption rates and were less sensitive to inhibition by oligomycin, an ATP synthase inhibitor. Copper-deficient and copper-adequate mitochondria responded similiarly to CCCP. No difference was observed in mitochondrial ATPase activity between copper-deficient and copper-adequate rats using submitochondrial particles. We conclude that cardiac mitochondrial respiration is compromised in copper-deficient rats, and may be related to an altered ATP synthase complex and/or a decreased mitochondrial membrane potential.     

The pH dependence of siliconiron interaction in rats

A 2 x 2 x 3 factorial experiment was conducted to study the pH dependence of a silicon-iron interaction in vivo. The dietary treatments used in the factorial design were the following (mg/kg of diet): silicon, 0 and 500; iron, 35 and 187; acid-base, ammonium chloride as 0.5% of total diet (acidic), sodium bicarbonate as 1.0% of total diet (basic), or no supplementation of acid or base (control). The supplementation of 500 mg silicon/kg of diet increased plasma-iron concentration in rats fed the acidic or control diets, but not in rats fed the basic diet. A high dietary-iron level suppressed copper absorption and utilization and subsequently imposed a negative effect on its own utilization. An increase in the plasma total-cholesterol concentration caused by high dietary-iron level was likely a consequence of the antagonistic effect of iron on copper absorption and utilization. The use of cupric sulfate pentahydrate as the dietary-copper source in this study resulted in plasma copper concentrations that were approximately twice those obtained in a related study using cupric carbonate. Also, a 42% coefficient of variation (C.V.) for plasma-copper concentrations of rats fed cupric sulfate in this study was greatly reduced from the C.V. = 108% previously associated with the dietary cupric carbonate.     

Role of plasma and serum proteases in the degradation of elastin

Accelerated proteolysis of tropoelastin and elastin occurs in the major arteries of chicks fed copper-deficient diets. Signs of elastin degradation are not obvious in normal arteries of copper-supplemented chicks. It is proposed that the sources of proteases that effect elastin degradation are from plasma and serum. Both calcium-dependent proteases and kallikrein were effective in degrading tropoelastin and partially crosslinked insoluble elastin into peptides similar to those detected in aortic extracts from copper-deficient chicks. As dietary copper deficiency progresses it is also possible to detect elastin peptides in plasma.