Increased rat brain cytochrome c correlates with degree of perinatal copper deficiency rather than apoptosis

Reductions in copper due to dietary restriction or transporter deficiency in brindled mice or humans with Menkes disease lead to reduced cuproenzyme activities, mitochondrial abnormalities, neurodegeneration and early mortality. The mechanisms for observed neuropathology remain unknown. Some researchers studying mutant mice suggest brain apoptosis as a possible factor based on changes in transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining and increased cytosolic cytochrome c and decreased Bcl-2 levels. Perinatal copper deficiency was induced in Holtzman rats during late gestation and lactation to investigate the role of apoptosis in the developing brain. Analysis of 13- and 24-d-old (P13 and P24) brains from male copper-deficient and copper-adequate rats revealed no difference in cytosolic cytochrome c or total Bcl-2 levels. Cerebellar TUNEL staining and caspase-3 activity were higher in the P12 copper-deficient than in the copper-adequate pups. However, TUNEL staining decreased and caspase-3 activity was not detected at P24 even though pups were more copper deficient based on cortex copper, Cu, Zn-superoxide dismutase and cytochrome c oxidase activities. This suggests that neuronal apoptosis is not enhanced by dietary copper deficiency in the brain. Lower Bcl-2 levels were detected in the copper-deficient rat hearts, consistent with apoptotic processes in mice reported by others. A robust enhancement of cytochrome c was observed in the total brain extracts and purified brain mitochondria of copper-deficient pups. Higher cytochrome c appeared to be correlated with the degree of copper deficiency and seemed to be associated with increased mitochondrial mass, because higher levels of voltage-dependent anion channel and mitochondrial complex I were also detected. The biochemical mechanisms for elevated cytochrome c are not known nor are the physiological consequences.     

Copper deficiency alters rat dopamine beta-monooxygenase mRNA and activity

Dopamine beta-monooxygenase (DBM), a cuproenzyme, converts dopamine to norepinephrine in selected cells. Studies were conducted in albino rats to resolve the known paradox of DBM after copper deficiency in which metabolite analyses of tissues suggest lower activity, whereas direct assay of homogenates suggests enhanced activity. After 4 wk of postweanling copper deficiency, male Holtzman rats exhibited 1.4-fold higher adrenal DBM activity and 1. 8-fold higher adrenal DBM mRNA levels than copper-adequate rats. Mixing experiments did not support the existence of endogenous activators or inhibitors. Adrenal catecholamine content indicated lower norepinephrine, higher dopamine and unaffected epinephrine content in copper-deficient compared with copper-adequate rats. Studies in 22-d-old male Sprague-Dawley offspring of dams started on copper deficiency at d 7 of gestation indicated similar results for adrenal DBM mRNA, a 1.75-fold increase compared with copper-adequate pups. Adrenal dopamine content was higher in female copper-deficient offspring compared with controls, but norepinephrine was not lower. Medulla oblongata/pons DBM mRNA concentration was higher in 22-d-old copper-deficient female but not male rats compared with controls. Six weeks of copper repletion to the 22-d-old rats restored adrenal DBM mRNA levels to control values. Enzyme assay and RNA results are consistent with enhanced formation of DBM in adrenal gland and noradrenergic cell bodies of copper-deficient rats. The molecular signal may not be solely lower norepinephrine content because adrenal DBM mRNA changes were evident in both nutritional models, whereas the norepinephrine content was altered only in the postnatal model.     

Calcium reintroduction decreases viability of cardiac myocytes from copper-deficient rats.

Copper deficiency leads to profound cardiac hypertrophy and failure. Myocytes were isolated from hearts of copper-deficient and copper-adequate male Holtzman rats to characterize size and function of the cells. Weanling rats were offered a semipurified diet low in copper in two separate experiments (Experiment 1, 0.45 mg Cu/kg and Experiment 2, 0.30 mg Cu/kg). Control (copper-adequate) rats drank water supplemented with cupric sulfate (20 mg Cu/L). Compared with copper-adequate rats, copper-deficient rats had lower hematocrits, liver copper concentrations and plasma ceruloplasmin activities, and higher heart weights and liver iron concentrations. When myocytes were isolated in low calcium media (1 micromol/L), cell viability was not affected by diet history. However, upon restoration to more physiologic levels of calcium (1 mmol/L), cells from copper-deficient rats were less viable, exhibiting an average loss of 34 and 40% in Experiments 1 and 2, respectively, compared with a 9.5 and 13% loss of cells, respectively, from the copper-adequate rats. Addition of the calcium channel blocker, verapamil, did not block this calcium-dependent loss of viability nor did the mitochondrial calcium channel blockers, ruthenium red and cyclosporin A. For comparison with another model of cardiac hypertrophy, the calcium sensitivity of myocytes from hypertrophic hearts of Sprague-Dawley rats with aortic constrictions was found not to differ from that of sham-operated rats. Thus, cardiac hypertrophy associated with postnatal copper deficiency results in a unique increased calcium intolerance of isolated myocytes.     

Serum cholesterol levels are not elevated in young copper-deficient rats, mice or brindled mice

Experiments were conducted with suckling male C57BL mice and Sprague-Dawley rats to investigate the relationship between copper deficiency and elevated serum cholesterol. Brindled mice, which have a genetic defect that affects copper distribution, were compared to their normal brothers. Dietary copper deficiency was produced in dams heterozygous for the brindled gene, in normal mouse dams and in rat dams. The subsequent male offspring were compared to those from copper-supplemented dams. Copper deficiency, as assessed by liver copper levels or ceruloplasmin activity, was demonstrated in 12-d-old rats, brindled mice, and in genotypically normal mice from dams fed the copper-deficient diet. However, serum cholesterol levels were not elevated in these "copper-deficient" rats or mice. In one experiment serum cholesterol levels of brindled mice were significantly lower than that of their littermate controls. An additional study was done with older mice. Their dams were fed a low copper diet from parturition throughout lactation, and the pups were fed the same copper-deficient diet for 4 wk after weaning. The 7-wk-old male copper-deficient mice had liver copper levels below 1 microgram/g, but no elevation in serum cholesterol was observed. The failure to demonstrate a rise in serum cholesterol in these perinatal models may be due in part to less severe hepatic copper deficiency because of neonatal copper reserves in liver.     

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.     

Effect of copper deficiency on the apolipoprotein E-rich high density lipoproteins in rats

Twenty-four male weanling Sprague-Dawley rats were randomly divided into two treatments, namely a copper-adequate (8 mg Cu/kg diet) or a copper-deficient (0.85 mg Cu/kg diet) group. Feed and distilled-demineralized water were provided ad libitum. After 7 wk, plasma high density lipoproteins (HDL) were isolated by ultracentrifugation and agarose column chromatography. A heparin-Sepharose affinity column chromatographic method was used to subfractionate HDL into apolipoprotein E (apoE) -rich and apoE-poor subfractions. Significantly higher protein and cholesterol contents of HDL and the second retained (RII) subfraction (apoE-rich) were observed in copper-deficient than in control rats. The apoE contents of HDL and the apoE-rich (RII) subfraction were about threefold higher in copper-deficient rats. The increase in apoE in the HDL fraction was due primarily to the increased apoE concentration of the RII subfraction. In addition to a higher apoE concentration, apolipoprotein A-I concentration was also higher in the RII subfraction of the copper-deficient rats as compared to controls. However, the percentage of these apolipoproteins remained unaltered by copper deficiency. The present data suggest that the hypercholesterolemia observed in copper deficiency was associated with marked increases in cholesterol and apolipoprotein contents of the apoE-rich HDL.     

Copper deficiency in a genetically hypertensive cardiomyopathic rat: electrocardiogram, functional and ultrastructural aspects

The effect of copper deficiency on cardiac function and structure was studied in a strain of rats (SHHS/Mcc-cp) known to develop cardiac failure as adults. Restriction of dietary copper (less than or equal to 1 mg/kg vs. 6 mg/kg in adequate diets) at weaning in both sexes for a 6-wk period produced cardiac hypertrophy. Male rats developed more severe copper-deficiency symptoms than their female counterparts. In both sexes of copper-deficient rats, there was an increase in cardiac length, width, free ventricular wall thickness and septum thickness. Electrocardiographic tracings revealed greater QRS height among male copper-deficient rats. Heart rate also was substantially reduced in this group. The increased volume of myocardium occupied by mitochondria in the copper-deficient male rats might result in increased electrical resistance that would increase the QRS height; hypertrophy or anemia also could be contributory. Some male copper-deficient rats had prolongation of the QRS in a bundle branch block pattern. Maximal rates of rise and fall for left ventricular pressure were reduced in male copper-deficient rats. The gross histology indicated that this type of heart failure was more concentric than eccentric. The copper-deficient male rat may serve as a useful model for studying the concentric cardiac hypertrophy that occurs in humans.     

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.     

Altered cytoskeletal organization and secretory response of thrombin-activated platelets from copper-deficient rats

The cytoskeletal organization that occurs following thrombin-induced activation of platelets was investigated in rats consuming either a copper-deficient diet containing less than 1 microgram Cu/g or a copper-adequate diet containing 5.5 micrograms Cu/g. Within 30 s following thrombin activation, the amount of polymerized actin in Triton X-100 extracts of whole platelets from copper-deficient rats was greater than the amount in platelets from copper-adequate rats. Electrophoretic analysis of the cytoskeletal proteins obtained from low speed centrifugation of the Triton X-100 extracts indicated that the myosin content of the cytoskeleton increased with time and reached higher levels following activation in platelets from copper-deficient rats. Actin content of the cytoskeleton also increased with time following activation. However, the difference in cytoskeletal actin content of platelets from copper-deficient and copper-adequate rats was not as great as that observed for myosin. The rate of ATP secretion from thrombin-activated platelets was also increased by copper deficiency. Myosin association with the platelet cytoskeleton may be involved in platelet secretion following thrombin activation. Thus, the increased association of myosin with the cytoskeleton and concomitant increase in ATP secretion suggest that the normal mechanism for stimulus-response coupling is altered in thrombin-activated platelets from copper-deficient rats.     

Myofibrillar and nonmyofibrillar myocardial proteins of copper-deficient rats

Myofibrillar and nonmyofibrillar proteins from hearts of copper-adequate (n = 9) and copper-deficient (n = 10) rats were compared. Male weanling Long-Evans rats were fed copper-deficient or copper-adequate diets for 9 wk. Twelve additional rats were fed similar diets and cardiac tissue was evaluated by transmission electron microscopy. Ventricular myocytes were glycerinated and homogenized in 0.1 M KCl and 1.5% Triton X-100, and suspensions were centrifuged at 1100 x g. The supernatant was removed and designated Triton X-100-soluble non-myofibrillar protein, and the pellet was resuspended and recentrifuged several times to obtain myofibrillar protein. Sodium dodecyl sulfate--polyacrylamide gel electrophoresis (SDS-PAGE) analysis was conducted on both protein fractions. Densitometer scans of SDS-PAGE pherograms of myofibrillar protein revealed no significant difference between copper-adequate and copper-deficient groups. Similar analysis of nonmyofibrillar protein revealed a consistent decrease or diminished level of a 23-kDa polypeptide among copper-deficient rat hearts. These results may be consistent with the findings that demonstrated fragementation of mitochondrial cristae and an increased area occupied by mitochondria in copper-deficient rat hearts.     

Lipid and fatty acid composition of organs from copper-deficient mice

Experiments were conducted to study the total lipid and fatty acid composition of liver, kidney, brain and heart of 7-wk-old male C57BL mice. Dietary copper deficiency was initiated at birth by feeding dams a purified diet containing 0.5 mg/kg copper. Offspring were fed the copper-deficient diet 4 wk postweaning. Control dams and offspring were fed the same diet but with added copper in the drinking water, 20 mg/L. Compared with controls the copper-deficient mice exhibited hepatomegaly, cardiac hypertrophy and a 4% reduction in brain weight as well as low ceruloplasmin activity (0.5% of control). Total phospholipid concentration in liver and kidney and total triacylglycerol concentration in kidney was lower in copper-deficient mice compared to concentrations measured in liver and kidney of control mice. The major change in essential fatty acid composition in the copper-deficient mice which was consistent between organs and lipid classes was a significantly lower proportion and absolute amount of dihomo-gamma-linolenic acid. Other changes in fatty acid composition were variable.     

Copper deficiency during perinatal development: effects on the immune response of mice

Dietary copper (Cu) was restricted in Swiss albino mice during five discrete intervals over a 9-wk period of perinatal development: gestation only (G), lactation only (L), 3 wk postlactation (PL), 1 wk after birth through postlactation (2/3L + PL), and lactation plus postlactation (L + PL). Biochemical and immunological status of mice in copper-deficient (-Cu) treatment groups in models G and L did not differ from that of copper-adequate (+Cu) controls. Signs of severe copper deficiency, such as low liver copper levels, and significant reductions in activity of plasma ceruloplasmin and splenocyte Cu-Zn superoxide dismutase were most evident in 6-wk-old mice from two groups, -Cu 2/3L + PL and -Cu L + PL. Mice in these groups were anemic and had small thymuses and enlarged spleens compared to controls receiving +Cu treatment. The -Cu mice demonstrated impaired antibody (plaque-forming cells, PFC) response to sheep erythrocytes, and the attenuation was proportional to copper deficiency, as judged by liver copper levels. Total plasma IgM levels were not greatly altered by -Cu treatment except in model L + PL. Total IgG levels were markedly reduced in this group and in the -Cu 2/3L + PL group. The PFC response of mice in the -Cu PL group was normal even though signs of copper deficiency were evident; however, the PFC response was reduced when -Cu treatment was extended to 5 wk and was reversible by switching to +Cu treatment. Splenocyte reactivity to B- and T-cell mitogens was not greatly different between groups. Incorporation of thymidine into DNA in the absence of mitogen was higher in -Cu mice. It is evident that severity of copper deficiency is related to degree of impaired immunity. Furthermore, severity of copper deficiency is dependent on duration and time of initiation of dietary copper restriction.     

Chronic dietary copper deficiency alters biochemical and morphological properties of mouse lymphoid tissues

Chronic copper deficiency in mice impairs both humoral and cell-mediated immunity, but the mechanisms are unknown. Copper deficiency was produced in C58 mice by feeding dams a diet low in copper throughout lactation and weaning the pups to this diet. Control mice were from dams fed the same diet but with copper supplementation the drinking water. Six-week-old mice were sampled for biochemical and morphological studies. Compared to copper-supplemented mice, copper-deficient animals were smaller, anemic and exhibited hypoceruloplasminemia. The copper-deficient mice have small thymus glands, enlarged spleens, and livers equivalent in size to copper-supplemented mice. Thymic atrophy is not caused by elevated serum corticosterone. Liver, spleen, and thymus tissues from copper-deficient mice exhibit low cytochrome oxidase (56, 38, and 45%, respectively) and superoxide dismutase activities (61, 60, and 43%, respectively) compared to tissues from copper-supplemented mice, indicating a functional copper deficiency. Electron micrographs taken of thymus and spleen from copper-deficient mice demonstrate altered morphology characterized by abnormal mitochondria and misshapen nuclei. Chronic copper deficiency alters the size, biochemistry and morphology of primary (thymus) and secondary (spleen) lymphoid tissue.     

Cardiac ultrastructural and electrophysiological abnormalities in postweanling copper-restricted and copper-repleted rats in the absence of hypertrophy.

Cardiac ultrastructural and functional characteristics were determined in copper-depleted and copper-repleted rats. Male weanling rats were randomized into five groups that were fed either copper-adequate or copper-deficient diets. After 5 wk, one group fed each diet was studied to obtain baseline values. At this time, one copper-adequate postweanling group continued to receive the adequate diet as control, one deficient postweanling group was fed the adequate diet to evaluate the effect of copper repletion and one postweanling adequate group was fed the deficient diet to evaluate copper depletion in relatively older rats. These dietary treatments were continued for six additional weeks. Copper-depleted rats of both ages exhibited significant cardiac ultrastructural pathology and electrocardiogram abnormalities and the postweanling copper-depleted rats exhibited these abnormalities in the absence of hypertrophy and anemia. Increased mitochondrial volume density, disarranged cristae, and nonaligned myofibrils with disturbances at Z-bands were displayed. Additionally, all copper-depleted rats demonstrated fragmented basal laminae at capillary-myocyte interface. Increased QRS amplitude and notching and greater QT intervals were displayed. Copper-repleted rats exhibited some, but not total, reversal of these abnormalities. These results suggest that capillary-myocyte interface changes may play an important role in the developing pathology of copper depletion.     

Copper deficiency alters plasma pool size, percent composition and concentration of lipoprotein components in rats.

Forty weanling male Sprague rats were randomly assigned to two dietary treatments, copper-deficient (9.0 mumol/kg diet) and copper-adequate (102.2 mumol/kg diet). After 7 wk of treatment, reductions in body weight and hematocrit, and an increase in relative heart weight, were observed in the copper-deficient rats. Plasma VLDL, LDL and HDL were isolated by ultracentrifugation and Sepharose column chromatography. In copper-deficient rats, the percent composition of protein was reduced by one-half, and triglyceride was increased by 1.6- and 2.7-fold in LDL and VLDL fractions, respectively. In VLDL, the marked increase in triglyceride was compensated by at least a 75% decrease in percent composition of cholesterol and phospholipids as a result of copper deficiency. No treatment difference in percent composition of HDL components was detected. When the data were expressed as the amount present in the vascular pool corrected for body weight, the plasma pool size of protein, triglyceride, phospholipid and cholesterol of LDL and HDL were increased twofold or more by copper deficiency. In VLDL, a sixfold increase in triglyceride, a 36% reduction in cholesterol, and no change in phospholipid and protein pool size were observed in copper-deficient rats. These data suggest that copper deficiency may enlarge the particle size but not particle number of VLDL, increase both particle size and number of LDL, and elevate particle number but not size of HDL.     

Female rats are protected against oxidative stress during copper deficiency

BACKGROUND: Copper deficiency induces a dramatic decrease of superoxide dismutase activity and leads to alteration of antioxidant defense systems. METHODS: and Objective: Experiments were conducted in weanling male, intact and ovariectomized female rats, fed either a copper-adequate or copper-deficient diet for seven weeks, in order to determine whether endogenous estrogen could modulate oxidative stress and the severity of copper-deficiency. RESULTS: Feeding male rats a copper-deficient diet induced typical signs of copper deficiency, such as decreased hepatic copper, growth retardation, anemia, heart hypertrophy, pancreas atrophy and hypercholesterolemia. Furthermore, copper deficiency increased the amount of lipid peroxidation products in the heart, liver and pancreas following in vitro iron induction. Although levels of hepatic copper in copper-deficient females were similar to those of their male counterparts, the females were partially protected from the adverse effects of the deficiency (no growth retardation, less severe anemia, lesser extent of lipid peroxidation). Thus, female rats are provided with a greater degree of protection against oxidative damage than males. However, females did not appear to be protected against pancreas atrophy, heart enlargement and hypercholesterolemia induced by copper deficiency. This observed partial protection of females was lost after ovariectomy as shown by decreased body weight and hematocrit, heart enlargement and higher tissue peroxidation in ovariectomized females compared to intact females. CONCLUSIONS: The results suggest that the partial protection of copper deficient females is related to the antioxidant properties of estrogens. The protective action of estrogen against oxidative stress is of particular importance when antioxidant defenses are decreased as shown in this experimental model.     

Mechanical properties of the copper-deficient rat heart

Copper deficiency is known to induce cardiac hypertrophy, cardiac morphologic lesions and altered electrocardiograms. These findings suggest that copper deficiency may also influence the mechanical properties of the myocardium. In the present study, weanling albino rats were fed a copper-deficient (-Cu) diet and compared to rats fed the same diet but with copper supplementation in the drinking water (+Cu). Rats were studied during a 1-week period following 4.5-5.5 weeks of treatment. When compared to +Cu rats, the -Cu rats exhibited characteristic signs of copper deficiency, such as reduced body weight, hypoceruloplasminemia, depressed hematocrit, low copper and elevated iron concentration in the liver. The -Cu rats also exhibited cardiac hypertrophy and both a dilution and depletion of left ventricular norepinephrine. Hearts were perfused and paced at both 27 degrees and 37 degrees. When compared to hearts from +Cu rats, the -Cu hearts: 1) had lower spontaneous heart rates, 2) had decreased coronary resistance, 3) gained significant weight during perfusion, 4) consumed more oxygen per unit pressure developed, and 5) developed less systolic pressure with a reduced rate of pressure development. However, the time to peak pressure development, one-halt relaxation time, and refractory period were not affected. The altered characteristics of the copper-deficient myocardium may be due to changes in the elastic properties of the muscle, aberrant energy metabolism or norepinephrine depletion.     

Decreased hephaestin activity in the intestine of copper-deficient mice causes systemic iron deficiency

Copper and iron metabolism intersect in mammals. Copper deficiency simultaneously leads to decreased iron levels in some tissues and iron deficiency anemia, whereas it results in iron overload in other tissues such as the intestine and liver. The copper requirement of the multicopper ferroxidases hephaestin and ceruloplasmin likely explains this link between copper and iron homeostasis in mammals. We investigated the effect of in vivo and in vitro copper deficiency on hephaestin (Heph) expression and activity. C57BL/6J mice were separated into 2 groups on the day of parturition. One group was fed a copper-deficient diet and another was fed a control diet for 6 wk. Copper-deficient mice had significantly lower hephaestin and ceruloplasmin (approximately 50% of controls) ferroxidase activity. Liver hepcidin expression was significantly downregulated by copper deficiency (approximately 60% of controls), and enterocyte mRNA and protein levels of ferroportin1 were increased to 2.5 and 10 times, respectively, relative to controls, by copper deficiency, indicating a systemic iron deficiency in the copper-deficient mice. Interestingly, hephaestin protein levels were significantly decreased to approximately 40% of control, suggesting that decreased enterocyte copper content leads to decreased hephaestin synthesis and/or stability. We also examined the effect of copper deficiency on hephaestin in vitro in the HT29 cell line and found dramatically decreased hephaestin synthesis and activity. Both in vivo and in vitro studies indicate that copper is required for the proper processing and/or stability of hephaestin.     

Heart murmurs, valvular regurgitation and electrical disturbances in copper-deficient genetically hypertensive, hypertrophic cardiomyopathic rats

OBJECTIVE: Rats with a genetic tendency to develop hypertensive, hypertrophic cardiomyopathy were fed copper-deficient diets and their cardiac responses were investigated. METHODS: Five male weanling rats of the Long-Evans and SHHF/Mcc-fa(cp) strains were randomly selected to receive diets containing either adequate quantities of copper (94.5 micromol Cu/kg diet) or reduced quantities of copper (<15.8 micromol Cu/kg diet) for 6 weeks, (n=5 within each group). Echocardiograms and electrocardiograms were recorded and analyzed at the end of the 6-week interval. RESULTS: Electrocardiograms from copper deficient groups showed longer Q-T intervals and increased QRS amplitudes than controls. Both the copper deficient and control SHHF groups demonstrated significant QRS complex prolongation compared to Long-Evans rats. Echocardiography analysis showed significant increases in left ventricular area, free wall dimension, and myocardial cross-sectional areas in rats fed a copper deficient diet. The frequency of systolic cardiac murmurs increased in copper deficient rats and were related to the presence of valvular regurgitation as determined from echocardiography. DISCUSSION: However, the data do not suggest that a copper-deficient diet fed to a strain of rats genetically susceptible to heart disease later in life, hastens or worsens the onset of cardiac disease. The genetic predisposition and copper-deficient states exert independent effects upon the heart.     

Hepatic cytochrome P-450 and microsomal heme oxygenase in copper-deficient rats

Copper deficient and control rats were pair-fed from weaning a milk-based diet. Each animal received 25 mg of iron parenterally. After 6 weeks of dietary manipulations the animals were killed. As expected, rats fed the copper-deficient diet were anemic. Copper-dependent enzyme activities and copper content of plasma and liver confirmed that copper deficiency was present in these rats. Hepatic microsomal cytochrome P-450 specific contents were similar in control and copper-deficient animals in the uninduced state and following induction of cytochrome P-450 by treatment with phenobarbital. However, microsomal heme oxygenase was increased in copper-deficient animals compared with controls whether they were treated with phenobarbital or not. No differences were observed in splenic microsomal heme oxygenase between dietary groups. These studies suggest that hepatic heme catabolism is enhanced in copper deficiency. The explanation for this enhancement may be related to increased hepatic iron accumulation in copper-deficient animals or to the associated defect of selenium metabolism in copper deficiency.