Copper Repletion Research Articles

Copper Deficiency Anemia Due to Low-Calorie, Blended Enteral Diet: Case Report.

Copper deficiency is an uncommon cause of hematologic abnormalities in children that is often overlooked or misdiagnosed. When found, specific recommendations on dose, duration, or frequency of copper repletion are lacking in the current literature. This report describes the case of a 4-year-old boy who presented to a university medical center in the midwestern United States with persistent macrocytic anemia found to be caused by copper deficiency secondary to a low-calorie, blended enteral diet. Intravenous copper supplementation was initiated at 40 μg/kg/d for a total of 6 days, followed by enteral supplementation of 2 mg cupric oxide. This repletion regimen ultimately improved the patient's copper deficiency and resolved anemia within 1 month.

Atherosclerotic lesion-specific copper delivery suppresses atherosclerosis in high-cholesterol-fed rabbits.

Dietary cholesterol supplements cause hypercholesterolemia and atherosclerosis along with a reduction of copper concentrations in the atherosclerotic wall in animal models. This study was to determine if target-specific copper delivery to the copper-deficient atherosclerotic wall can block the pathogenesis of atherosclerosis. Male New Zealand white rabbits, 10-weeks-old and averaged 2.0 kg, were fed a diet containing 1% (w/w) cholesterol or the same diet without cholesterol as control. Twelve weeks after the feeding, the animals were injected with copper-albumin microbubbles and subjected to ultrasound sonication specifically directed at the atherosclerotic lesions (Cu-MB-US) for target-specific copper delivery, twice a week for four weeks. This regiment was repeated 3 times with a gap of two weeks in between. Two weeks after the last treatment, the animals were harvested for analyses of serum and aortic pathological changes. Compared to controls, rabbits fed cholesterol-rich diet developed atherosclerotic lesion with a reduction in copper concentrations in the lesion tissue. Cu-MB-US treatment significantly increased copper concentrations in the lesion, and reduced the size of the lesion. Furthermore, copper repletion reduced the number of apoptotic cells as well as the content of cholesterol and phospholipids in the atherosclerotic lesion without a disturbance of the stability of the lesion. The results thus demonstrate that target-specific copper supplementation suppresses the progression of atherosclerosis at least in part through preventing endothelial cell death, thus reducing lipid infiltration in the atherosclerotic lesion.

Copper Deficiency

BACKGROUND: Acquired copper deficiency in the pediatric population is a rare event but given the hematologic and potentially irreversible neurologic consequences, prompt recognition and treatment is important. Copper is an essential cofactor in enzymatic reactions essential to proper hematologic, skeletal, neurologic and vascular function. Copper is found in a variety of foods including meats, nuts, legumes, and whole grains while lacking in highly processed foods. Copper requirements in children over the age of 4 is 15 mg/day, which is readily acquired in a typical diet. Copper deficiency is known to occur in patients with the rare X-linked mutation in the ATP7A copper transport protein, known as Menkes Syndrome, and in older individuals with gastrointestinal bypass surgery who lose the ability to absorb copper through the duodenum and proximal jejunum, however, it is rarely reported in other conditions. Pediatric patients with autism spectrum disorders or developmental delay with a limited dietary repertoire are at risk for copper deficiency thus a high index of suspicion must exist in order to diagnose the disorder.CASE PRESENTATION: A 15 y/o boy with a prior diagnosis of global developmental delay and oral aversion presented with parental concerns for worsening fatigue, weakness and gait instability. Symptoms had been slowly progressive and accompanied by weight loss. His longstanding feeding difficulties had been refractory to intensive feeding programs yet he continued to feed orally (with no prior attempts at enteral/parenteral nutrition). A daily diet consisted only of 50-60 oz of milk and 25-30 individual servings of butterscotch pudding (1680-1880 calories/day, 0.7mg iron/day).Upon presentation he was afebrile with global muscle weakness. Growth parameters were at the 25th and 2nd percentiles for height and weight, respectively. Initial complete blood count demonstrated white blood cell count of 3.3, absolute neutrophil count of 760, hemoglobin of 4.4 with MCV of <50, reticulocyte count of 0.5, platelet count of 392. Review of his peripheral blood smear revealed microcytic, hypochromic red cells without marked fragmentation, anisopoikilocytosis or ringed sideroblasts; there were no morphologic abnormalities of his leukocytes or platelets. Iron studies demonstrated ferritin of 45, total iron binding capacity of 514, and 2% iron saturation. He had no evidence of B12 or folate deficiency. Stool guaiac was negative for occult blood loss. He was hospitalized to receive transfusion of red blood cells and begin IV iron therapy due to his inability to tolerate oral repletion.Over the the next 2 months he demonstrated some improvement in his anemia but had worsening leukopenia, neutropenia and a declining platelet count. He had worsening neurologic function with increasing need for assistance in his activities of daily living. Due to lack response to IV iron and worsening cytopenias, additional evaluation was performed which revealed a serum copper level of 6 (range 60-190), and cerulosplasmin of 2.1 (range 22-58).Once a diagnosis of copper deficiency was made, the patient promptly began a course of parenteral copper repletion. He received IV copper 35mcg/kg/day x 3 days then weekly intravenous infusions. Given his malnutrition, a G-tube was placed to begin oral copper repletion and enteral nutrition. Within 3 weeks his copper level improved as well as his blood counts. Unfortunately, although his blood counts and copper levels normalized, his neurologic status remains below his old baseline although he has made gains in his gross and fine motor abilities.DISCUSSION: Children with limited dietary repertoires are at risk for otherwise rare nutritional deficiencies such as copper deficiency. It is not uncommon that nutritional deficiencies may become more prominent when children reach puberty and have increased dietary requirements not only of calories, but also of micronutrients and trace elements. This patient's clinical course demonstrates the need to have a high index of suspicion of concomitant nutritional deficiencies other than those routinely evaluated such as iron, B12 and folate given his finding of both iron and copper deficiency. Review of his blood smear at presentation was consistent with iron deficiency and did not demonstrate typical hallmarks of copper deficiency such as ringed sideroblasts. DisclosuresMalec:Bioverativ: Consultancy, Other: Research funding; Shire: Consultancy, Other: Research funding.

Active uptake of hydrophilic copper complex Cu(ii)-TETA in primary cultures of neonatal rat cardiomyocytes.

Myocardial ischemia leads to copper efflux from the heart. The ischemic tissue with a low copper content fails to take up copper from the circulation even under the conditions of serum copper elevation. Cardiac copper repletion thus requires other available forms of this element than those currently known to bind to copper transport proteins. The copper complex of triethylenetetramine (TETA) is a metabolite of TETA, which has the potential to increase cardiac copper content in vivo. In the present study, we synthesized Cu(ii)-TETA, analyzed its crystal structure, and demonstrated the role of this compound in facilitating copper accumulation in primary cultures of neonatal rat cardiomyocytes. The Cu(ii)-TETA compound formed a square pyramidal chloride salt [Cu(TETA)Cl]Cl structure, which dissociates from chloride in aqueous solution to yield the four-coordinate dication Cu(ii)-TETA. Cu(ii)-TETA was accumulated as an intact compound in cardiomyocytes. Analysis from time-dependent copper accumulation in cardiomyocytes defined a different dynamic process in copper uptake between Cu(ii)-TETA and CuCl2 exposure. An additive copper accumulation in cardiomyocytes was found when the cells were exposed to both CuCl2 and Cu(ii)-TETA. Gene silencing of copper transport 1 (CTR1) did not affect cross-membrane transportation of Cu(ii)-TETA, but inhibited copper cellular accumulation from CuCl2. Furthermore, the uptake of Cu(ii)-TETA by cardiomyocytes was ATP-dependent. It is thus concluded that the formation of Cu(ii)-TETA facilitates copper accumulation in cardiomyocytes through an active CTR1-independent transportation process.

Regression of pressure overload‐induced cardiac hypertrophy by TETA‐mediated myocardial copper supplementation in rats

Sustained pressure overload leads to cardiac hypertrophy and copper efflux from the heart. Dietary copper supplementation replenishes copper in the heart and reverses cardiac hypertrophy. On the contrary, a copper selective chelator, trientine (TETA), reverses left ventricular hypertrophy associated with diabetes. The present study was undertaken to test the hypothesis that TETA functions as a copper chaperone delivering copper to the heart leading to regression of cardiac hypertrophy. Adult male Sprague‐Dawley rats were subjected to transverse aortic constriction (TAC) to induce cardiac hypertrophy. Eight weeks after the surgery cardiac hypertrophy was developed and myocardial copper was reduced. TETA was then administrated by gavage at two different doses (21.9 mg/kg·day and 87.6 mg/kg·day) for six weeks. The results showed that at lower dose, TETA replenished copper contents in the heart, accompanied by a decrease in blood plasma and an increase in urine of copper concentrations. At higher dose, TETA did not replenish copper contents in the heart, but markedly increased copper concentrations in the urine, along with a decrease in serum copper concentrations. Corresponding to myocardial copper repletion, lower dose TETA reversed cardiac hypertrophy, as judged by a reduction in the left ventricle wall thickness (LVAWd and LVPWd) and a decrease in the heart size (heart weight/tibia length), and diminished cardiac fibrosis, as reflected by a decrease in collagen I content. TETA at higher dose not only did not regressed cardiac hypertrophy, but caused cardiac hypertrophy in sham‐operated rats. This study demonstrates that TETA, at lower dose, functioned as a copper chaperone delivering copper to the heart of copper deprivation and, at higher dose, simply remained its chelator function removing copper from the body by urinary excretion.Support or Funding InformationSupported by National Science Foundation of China (81230004 and 81300109).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The Involvement of Cytochrome c Oxidase in Mitochondrial Fusion in Primary Cultures of Neonatal Rat Cardiomyocytes.

Cytochrome c oxidase (CCO) is a copper-dependent enzyme of mitochondrial respiratory chain. In pressure overload-induced cardiac hypertrophy, copper level and CCO activity are both depressed, along with disturbance in mitochondrial fusion and fission dynamics. Copper repletion leads to recovery of CCO activity and normalized mitochondrial dynamics. The present study was undertaken to define the link between CCO activity and mitochondrial dynamic changes. Primary cultures of neonatal rat cardiomyocytes were treated with phenylephrine to induce cell hypertrophy. Hypertrophic cardiomyocytes were then treated with copper to reverse hypertrophy. In the hypertrophic cardiomyocytes, CCO activity was depressed and mitochondrial fusion was suppressed. Upon copper repletion, CCO activity was recovered and mitochondrial fusion was reestablished. Depression of CCO activity by siRNA targeting CCO assembly homolog 17 (COX17), a copper chaperone for CCO, led to fragmentation of mitochondria, which was not recoverable by copper supplementation. This study thus demonstrates that copper-dependent CCO is critical for mitochondrial fusion in the regression of cardiomyocyte hypertrophy.

Optic Neuropathy, Myelopathy, Anemia, and Neutropenia Caused by Acquired Copper Deficiency After Gastric Bypass Surgery

Malabsorptive bariatric surgery is rapidly becoming a major cause of copper deficiency given the increasing prevalence of these procedures for morbid obesity. Acquired copper deficiency can present with clinically significant hematologic and neurological manifestations. Although hematologic manifestations of copper deficiency are rapidly reversible, significant neurological improvement after copper supplementation therapy is unusual and many patients remain debilitated and may only experience, at best, stabilization of the neurological manifestations. Here we present a case of an undiagnosed copper deficiency several years after bariatric gastric bypass surgery, in a patient who concomitantly used zinc-containing denture cream for several years, associated with anemia, neutropenia, myelopathy, respiratory failure, and bilateral optic neuropathy, which caused major vision loss. This patient was also a heterozygote carrier of the 5,10-methylenetetrahydrofolate reductase A1298C gene polymorphism, which may affect copper metabolism. Intravenous copper repletion resulted in rapid correction of hematologic indices. However, neurological manifestations, including vision loss responded only modestly to copper supplementation, despite achieving normal blood copper concentrations. Clinicians should consider copper deficiency in patients at risk, as in this case, as a delayed diagnosis can lead to irreversible disability due to neurological manifestations.

Rapid alteration in rat red blood cell copper chaperone for superoxide dismutase after marginal copper deficiency and repletion

There is increased incidence of human copper deficiency (CuD). A sensitive and reliable blood biomarker may reveal additional cases of marginal deficiency. Two experiments were designed to test the hypothesis that the copper chaperone for superoxide dismutase (CCS) would be a robust marker after marginal CuD. Experiment 1 used weanling male Sprague-Dawley rats that were offered a CuD diet for 4 weeks, and samples were evaluated after 1, 2, and 4 weeks and compared with copper-adequate (CuA) controls. Furthermore, iron-deficient rats were included for comparison after 2 weeks of depletion. Red blood cell and plasma cuproenzymes were evaluated through Western blot analysis. Superoxide dismutase (Sod1) and ceruloplasmin protein were found to be altered by both iron and CuD, whereas CCS and CCS/Sod1 ratio were found to only be altered only in CuD rats and, importantly, after only 1 week of treatment. Two weeks on CuA diet restored cuproenzyme levels to control values after 4 weeks of CuD depletion. In experiment 2, marginal CuD (CuM) rats were compared with CuA and CuD rats after 2 weeks of treatment. Superoxide dismutase, ceruloplasmin, and CCS/Sod1 abundances were lower in CuM and CuD groups compared with CuA rats, but there was no statistical difference between CuM and CuD rats. However, CCS was statistically different between all groups, and abundance highly correlated with liver copper concentration. Results suggest that red blood cell CCS may be an excellent biomarker for diagnosis of rapid and marginal CuD.

Acquired Copper Deficiency: A Potentially Serious and Preventable Complication Following Gastric Bypass Surgery

Copper is an essential cofactor in many enzymatic reactions vital to the normal function of the hematologic, vascular, skeletal, antioxidant, and neurologic systems. Copper deficiency in the United States is believed to be relatively rare but has been described in the setting of zinc supplementation, myelodysplastic syndrome, use of parenteral nutrition and chronic tube feeding, and in various malabsorptive syndromes, including following gastrectomy and gastric bypass surgery. Features of copper deficiency include hematologic abnormalities (anemia, neutropenia, and leukopenia) and myeloneuropathy; the latter is a rarer and often unrecognized complication of copper deficiency. We here describe two patients who presented with severe gait abnormalities and anemia combined with neutropenia several years after roux-en-Y gastric bypass (RYGB) surgery for obesity who were found to be severely copper deficient. Intravenous copper repletion resulted in the rapid correction of hematologic indices; combined intravenous and oral copper supplementation and eventual oral copper supplements alone normalized serum copper levels in each patient, but resulted in only partial resolution of the neurologic deficits. This report serves to alert physicians of the association between RYGB procedures and subsequent copper deficiency in order to avoid diagnostic delays and to improve treatment outcomes.

Cu‐repletion promotes angiogenesis in the Cu‐deficient rat heart

Heart hypertrophy is a common manifestation of dietary copper deficiency in experimental animals. Upon repletion of copper, the hypertrophy is quickly reversed. Knowing this, the aim of this study was to compare angiogenesis during the hypertrophy and reverse remodeling seen in copper deficiency/repletion. Male adult rats (225 g) were fed either a purified Cu-adequate diet (6 mg Cu/kg diet; n=3) or a Cu-deficient diet (0.3 mg Cu/kg diet; n=4) for 8 weeks. Cu-deficient animals were then replenished with Cu-adequate diet for 1, 2 or 4 weeks (n= 3 adequate and 4 repleted for each). Echocardiography and heart wt/body wt ratio were used to determine hypertrophy. Heart tissue samples were collected and stained with anti-Factor VIII to quantify angiogenesis. Vascular endothelial growth factor (VEGF) was assayed by Western blot. Cu-deficient hearts exhibited significant hypertrophy along with lower capillary density and reduced VEGF content. Cu-repletion caused coincident increases in VEGF and angiogenesis. With repletion, the hypertrophy indices returned to control values. These results demonstrate an inverse relationship between angiogenesis and heart hypertrophy. The findings suggest involvement of a VEGF-dependent mechanism in myocardial reverse remodeling during copper repletion. Supported by NIH grants DK055030, HL59225 and HL63760.

Cardiac Cytochrome-c Oxidase Deficiency Occurs During Late Postnatal Development in Progeny of Copper-Deficient Rats

Although cytochrome-c oxidase (CCO) is a copper-dependent enzyme, the effect of maternal copper deficiency on the expression of CCO activity during postnatal development of the neonatal rat heart has not been investigated extensively. Here, we show that CCO activity in heart mitochondria isolated from neonates of copper-deficient dams did not exhibit significant reductions until postnatal days (PND) 15 and 21. In addition, immunoblot analysis indicated that the CCO subunit (Cox-1) was reduced on postnatal Days 10 and 21, and that Cox-4 was reduced on PND 21 in heart mitochondria of the neonates from copper-deficient dams. These findings indicate that the impairment of CCO activity in neonatal heart by maternal copper deficiency occurs late in the postnatal heart development. Furthermore, the concurrent reductions in Cox-1 and Cox-4 suggest that the impaired CCO activity reflects a CCO deficiency in heart mitochondria. CCO activity and Cox-1 in heart mitochondria were not fully restored by 6 weeks of postweaning copper repletion in the pups of copper-deficient dams. This indicates that prolonged maternal intake of moderately low dietary copper produces CCO deficiency in cardiac mitochondria of neonates during late postnatal heart development, after terminal differentiation of cardiomyocytes occurs. The resistance of CCO deficiency to repair by dietary copper supplementation may be related to the relatively slow turnover of the affected mitochondria in the terminally differentiated heart.

Phase 2 trial of copper depletion and penicillamine as antiangiogenesis therapy of glioblastoma

Penicillamine is an oral agent used to treat intracerebral copper overload in Wilson's disease. Copper is a known regulator of angiogenesis; copper reduction inhibits experimental glioma growth and invasiveness. This study examined the feasibility, safety, and efficacy of creating a copper deficiency in human glioblastoma multiforme. Forty eligible patients with newly diagnosed glioblastoma multiforme began radiation therapy (6000 cGy in 30 fractions) in conjunction with a low-copper diet and escalating doses of penicillamine. Serum copper was measured at baseline and monthly. The primary end point of this study was overall survival compared to historical controls within the NABTT CNS Consortium database. The 25 males and 15 females who were enrolled had a median age of 54 years and a median Karnofsky performance status of 90. Surgical resection was performed in 83% of these patients. Normal serum copper levels at baseline (median, 130 microg/dl; range, 50-227 microg/dl) fell to the target range of <50 microg/dl (median, 42 microg/dl; range, 12-118 microg/dl) after two months. Penicillamine-induced hypocupremia was well tolerated for months. Drug-related myelosuppression, elevated liver function tests, and skin rash rapidly reversed with copper repletion. Median survival was 11.3 months, and progression-free survival was 7.1 months. Achievement of hypocupremia did not significantly increase survival. Although serum copper was effectively reduced by diet and penicillamine, this antiangiogenesis strategy did not improve survival in patients with glioblastoma multiforme.

Phase II trial of copper depletion as angiosuppressive treatment in newly diagnosed Glioblastoma Multiforme: Final report

1530 Background: Penicillamine is an oral agent used to treat intracerebral copper overload in Wilson's disease. Copper is a known regulator of angiogenesis; copper reduction inhibits experimental glioma growth and invasiveness. This study examined the feasibility, safety, and efficacy of copper deficiency in human glioblastoma multiforme. Methods: Forty eligible patients with newly diagnosed glioblastoma began concurrent radiation therapy (6000 cGy) in conjunction with a low copper diet and gradual dose escalation of penicillamine. Serum copper was measured at baseline and monthly. The primary endpoint was overall survival compared to controls in the NABTT database. Results: Twenty-five males and 15 females were enrolled between March 1999 and April 2000. The median age was 54 years, the mean KPS was 88, and 83% received surgical debulking. The mean baseline serum copper level was 138.6 ± 31.6 μg/dL and generally fell to the target range of 50–70 μg/dL (57.5 ± 27.5) after two months. Hypocupremia was found to be well tolerated for months. Significant toxicities such as myelosuppression, elevated liver function values, and skin rash were rapidly reversed by copper repletion. The median survival was 11.3 months and the median progression-free survival was 7.1 months. Prolonged survival was related to age and extent of surgical resection, but not hypocupremia. Conclusions: Although serum copper was effectively reduced by diet and penicillamine in patients with newly diagnosed glioblastoma multiforme who also received radiation, this antiangiogenesis strategy did not improve survival. No significant financial relationships to disclose.

Phase II trial of copper depletion as angiosuppressive treatment in newly diagnosed Glioblastoma Multiforme: Final report

1530 Background: Penicillamine is an oral agent used to treat intracerebral copper overload in Wilson's disease. Copper is a known regulator of angiogenesis; copper reduction inhibits experimental glioma growth and invasiveness. This study examined the feasibility, safety, and efficacy of copper deficiency in human glioblastoma multiforme. Methods: Forty eligible patients with newly diagnosed glioblastoma began concurrent radiation therapy (6000 cGy) in conjunction with a low copper diet and gradual dose escalation of penicillamine. Serum copper was measured at baseline and monthly. The primary endpoint was overall survival compared to controls in the NABTT database. Results: Twenty-five males and 15 females were enrolled between March 1999 and April 2000. The median age was 54 years, the mean KPS was 88, and 83% received surgical debulking. The mean baseline serum copper level was 138.6 ± 31.6 μg/dL and generally fell to the target range of 50–70 μg/dL (57.5 ± 27.5) after two months. Hypocupremia was found to be well tolerated for months. Significant toxicities such as myelosuppression, elevated liver function values, and skin rash were rapidly reversed by copper repletion. The median survival was 11.3 months and the median progression-free survival was 7.1 months. Prolonged survival was related to age and extent of surgical resection, but not hypocupremia. Conclusions: Although serum copper was effectively reduced by diet and penicillamine in patients with newly diagnosed glioblastoma multiforme who also received radiation, this antiangiogenesis strategy did not improve survival. No significant financial relationships to disclose.

Dietary Copper Restriction-Induced Changes in Myocardial Gene Expression and the Effect of Copper Repletion

Dietary copper (Cu) restriction leads to cardiac hypertrophy and failure in mice, and Cu repletion (CuR) reverses the hypertrophy and prevents the transition to heart failure. The present study was undertaken to determine changes in myocardial gene expression involved in Cu deficient (CuD) cardiomyopathy and its reversal by CuR. Analysis was performed on three groups of mice: 4-week-old CuD mice that exhibited signs of cardiac failure, their age-matched copper-adequate (CuA) controls, and the CuD mice that were re-fed adequate Cu for 2 weeks. Total RNA was isolated from hearts and subjected to cDNA micro-array and real-time reverse transcription-polymerase chain reaction analysis. Dietary CuD caused a decrease in cardiac mRNA of beta-MHC, L-type Ca(2+) channel, K-dependent NCX, MMP-2, -8, and -13, NF-kappaB, and VEGF. The mRNA levels of ET-1, TGF-beta, TNF-alpha, and procollagen-I-alpha1 and III-alpha1 were increased in the CuD cardiac tissue. Copper repletion resulted in cardiac mRNA levels of most of the genes examined returning to control levels, although the K-dependent NCX and MMP-2 values did not reach those of the CuA control. In addition, CuR caused an increase in beta-MHC, L-type Ca(2+)channel, MMP-13 to levels surpassing those of CuA control, and a decrease in ET-1, and TNF-alpha mRNA levels. In summary, changes in gene expression of elements involved in contractility, Ca(2+) cycling, and inflammation and fibrosis may account for the altered cardiac function found in CuD mice. The return to normal cardiac function by CuR may be a result of the favorable regression in gene expression of these critical components in myocardial tissue.

Regression of Dietary Copper Restriction-Induced Cardiomyopathy by Copper Repletion in Mice

Dietary copper deficiency (CuD)(3) leads to cardiac hypertrophy in various animal models. We showed recently that heart failure develops after hypertrophy in FVB mice fed a CuD diet. The present study was undertaken to determine whether CuD-induced cardiac failure is reversible upon copper repletion (CuR). Dams of FVB mice were fed a CuD diet (0.3 mg/kg) starting from d 3 postdelivery; the weanling pups were fed the same diet until CuR with 6.0 mg/kg Cu in the diet at 4 or 5 wk of age. CuR at 4 wk of age prevented the body weight loss; at 5 wk of age, it resulted in the regaining of the lost weight caused by CuD. A significant regression of CuD-induced cardiac hypertrophy was observed in the CuR mice. Histopathological examination revealed that CuR eliminated CuD-caused lipid deposition in the myocardium, and electron microscopy demonstrated that CuD-induced ultrastructural changes such as mitochondrial swelling and organelle structural disarray were all reversed in the CuR mice. Hemodynamic analysis showed that the CuD-depressed systolic and diastolic parameters such as the maximal rate of left ventricular pressure rise (+dP/dt) and decline (-dP/dt), and the contraction and relaxation times were completely recovered in the CuR mice. Furthermore, the CuD-blunted myocardial responses to the beta-adrenergic agonist, isoproterenol, were also restored in the CuR mice. This study thus demonstrates for the first time that CuR results in the regression of heart failure induced by CuD as demonstrated by the reversal of depressed cardiac hemodynamic and contractile function and the restored responsiveness to beta-adrenergic stimulation.

Effect of Copper Source and Level on the Rate and Extent of Copper Repletion in Holstein Heifers

The objective of this study was to evaluate the rate and extent of Cu repletion in Holstein heifers using two Cu sources (organic and inorganic) at two levels (15 and 30mg/kg). An additional repletion treatment included a Cu oxide bolus. Heifers (n = 50) were individually fed a total mixed ration fortified with S and Mo at 0.40%, and 15mg/kg of dry matter of the total diet, respectively. After 111 d of depletion, heifers were stratified by liver Cu concentration and randomly allotted to one of five repletion treatments. Four treatments consisted of feed sources of Cu (feed-Cu), 1) CuSO4 at 15mg/kg; 2) CuSO4 at 30mg/kg; 3) Availa-Cu at 15mg/kg; and 4) Availa-Cu at 30mg/kg. Availa-Cu is an organic Cu source that produces a Cu-amino acid complex. A fifth treatment, consisting of an intraruminal bolus (IB), provided a single dose of 25g of CuO needles. Repletion treatments were delivered in the same total mixed ration without supplemental S and Mo. Copper status was assessed in blood and liver samples collected on 14-d intervals for 70 d. Irrespective of treatment, all heifers increased in body weight during the repletion period. Liver Cu increased in each feed-Cu treatment over time. Heifers treated with an IB reached a peak in liver Cu concentration (165.5mg/kg) on d 28. Mean liver Cu concentrations were higher in heifers receiving 30mg/kg of Cu compared with heifers receiving 15mg/kg of Cu. Red blood cell superoxide dismutase (SOD) activity was higher (P<0.001) in heifers receiving CuSO4 than Availa-Cu (0.98 vs 0.87 U). Also, SOD activity was higher when heifers were supplemented with 30 vs 15mg/kg Cu (0.98 vs 0.87 U). Heifers receiving the Cu IB had higher SOD activity than heifers receiving feed-Cu sources (1.03 vs 0.92 U). Plasma ceruloplasmin concentration was higher (P<0.001) in IB-treated heifers vs. other treatments. No differences in plasma ceruloplasmin were detected for feed-Cu source or level. These results indicate that all Cu sources evaluated in this study elevated Cu status of depleted heifers, particularly when provided at higher dietary levels.

Copper repletion enhances apical iron uptake and transepithelial iron transport by Caco-2 cells.

The influence of copper status on Caco-2 cell apical iron uptake and transepithelial transport was examined. Cells grown for 7-8 days in media supplemented with 1 microM CuCl(2) had 10-fold higher cellular levels of copper compared with control. Copper supplementation did not affect the integrity of differentiated Caco-2 cell monolayers grown on microporous membranes. Copper-repleted cells displayed increased uptake of iron as well as increased transport of iron across the cell monolayer. Northern blot analysis revealed that expression of the apical iron transporter divalent metal transporter-1 (DMT1), the basolateral transporter ferroportin-1 (Fpn1), and the putative ferroxidase hephaestin (Heph) was upregulated by copper supplementation, whereas the recently identified ferrireductase duodenal cytochrome b (Dcytb) was not. These results suggest that DMT1, Fpn1, and Heph are involved in the iron uptake process modulated by copper status. Although a clear role for Dcytb was not identified, an apical surface ferrireductase was modulated by copper status, suggesting that its function also contributes to the enhanced iron uptake by copper-repleted cells. A model is proposed wherein copper promotes iron depletion of intestinal Caco-2 cells, creating a deficiency state that induces upregulation of iron transport factors.

Copper Deficiency Alters Rat Dopamine β-Monooxygenase mRNA and Activity

Dopamine β-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.

Copper Status Affects Immune Response by Modulating Macrophage Response to Stimuli

LEARNING OUTCOME: To compare the effect of copper depletion and repletion on one aspect of immune response: macrophage response to stimuli in vitro. INTRODUCTION: Copper is an essential trace element for animals and humans. Cardiovascular pathology due to copper deficiency is well-documented in animals and humans with genetic disease, but the effect of copper status on immune function needs elucidation. METHODS: Cells from the murine macrophage cell line RAW264.7 were cultured in Dulbecco's minimal essential medium with Earle's salts and bovine serum. Tetraethylpentamine (TEPA), 0-20 μM, was added to chelate copper. Copper repletion was achieved by adding CuCl 2, 0-10 μM. Cells were stimulated with interferon-γ (5U/mL) and bacterial lipopolysaccharide (100ng/mL). Cell viability was determined by dye reduction. Nitric oxide produced was measured as its stable degradatory product, nitrite, by the Griess reaction. Casein-induced macrophages from mice were similarly assayed. RESULTS: Increasing copper levels consistently elevated nitrite production in stimulated RAW264.7 (maximal increase at 16 μM, 48.6±2.7%) and macrophages (maximal increase at 10 μM, 90.8±4.6%), whereas chelating copper with TEPA decreased nitrite production in RAW264.7 (maximal reduction at 8 μM, 26.7±1.8%) and macrophages (maximal reduction at 10 μM, 27.8%). Differences were significant at P=0.001. CONCLUSION: Since macrophages are pivotal components of the immune system, its modulation by copper status has important implications for host resistance to infections, neoplasm's and patients with AIDS. Further research is needed to elucidate the effect of copper status on immunity in vivo.