Progress 07/21/04 to 01/22/09
Outputs
Progress Report Objectives (from AD-416) Overall, to determine, using animal models, whether copper (Cu) intakes consistent with those observed in humans can adequately support cardiovascular functions. To develop a strategy for assessment of marginal copper deficiency in animals; to use this strategy to determine biomarkers of copper status that are suitable for assessment of marginal status in humans. To determine the contribution of oxygen- and nitrogen- derived reactive species to the cardiomyopathy (metabolic, contractile) induced by Cu deficiency, and the dietary intakes at which this pathology occurs. To determine whether low Cu intakes consistent with those observed in humans can impair nitric oxide-dependent control of blood vessels and blood pressure regulation. To determine whether the oxidative stress induced by Cu deficiency affects homocysteine metabolism and, thereby, cardiovascular function, and whether such effects influence nitric oxide-dependent signal transduction and/or other mechanisms that affect atherosclerosis. To determine whether marginal Zn deficiency can exacerbate or unmask cardiovascular effects of sub-optimal Cu status by virtue of its role in oxidative/nitrosative metabolism. Approach (from AD-416) Laboratory animals of varying ages and, in some cases, varying genetic makeup will be fed diets containing copper in severely deficient, marginally deficient or adequate amounts for varying periods of time. Based on comparisons to reliable invasive markers of copper status, non- invasive biomarkers will be tested for validity in assessing marginal copper deficiency. Tests of cardiovascular function and examinations of mechanism of depressed function will be made over ranges of copper status varying from severely-deficient to adequate. Cardiovascular functional measurements include heart contractile function, cardiac mitochondrial respiration and respiratory complex activity, blood vessel contractility and blood pressure. Atherosclerosis will be assessed by microscopic observation of blood vessels. Examination of mechanisms of depressed function will focus on the effects of oxidative stress and altered nitric oxide metabolism known to occur in copper deficiency. Such examination will include assessment of oxidative/nitrosative damage, altered nitric oxide signaling and altered homocysteine metabolism and will extend to measurement of transcription factors, mRNA and enzymes that influence and are influenced by oxidative stress and nitric oxide signaling. Significant Activities that Support Special Target Populations During the 5 years of this project, progress was made in identifying mechanisms for the effects of Cu deficiency on cardiovascular function and identifying low copper intake during pregnancy as a factor for cardiovascular disease in offspring. Research findings showed that hearts of copper-deficient mice have reduced contractile pressure, elevated relaxation pressure, impaired responsiveness to adrenalin excitation, and increased collagen deposition. These changes in the heart are characteristic of heart failure and show that dietary Cu deficiency is a risk factor for heart disease and subsequent cardiac failure.Furthermore, characteristics of heart failure were found in adult rats even at marginally low Cu intake. In terms of vascular function, findings from this project showed that increased blood pressure caused by copper deficiency in rats was caused by the attenuation of vascular relaxation resulting from decreased levels of the vascular relaxant, nitric oxide. It was also shown that copper deficiency promotes vascular inflammation independently of its effect on nitric oxide levels. These findings indicate that low dietary copper intake increases the risk for cardiovascular disease by promoting hypertension and vascular inflammation. Other studies performed in this project indicate that dietary copper may be important for reversing some forms of heart disease. In mice, surgical constriction of the aorta produces chronic pressure overload and eventual cardiac enlargement and failure. It was found that that copper supplementation of the mice having constricted aortas reversed the cardiac enlargement and prevented heart failure by promoting the formation of blood vessels in the heart. These findings indicate that copper requirements may increase in patients with certain types of heart disease associated with cardiac enlargement and that copper supplementation may be beneficial for improving their cardiac function. Studies with pregnant rats showed that low copper intakes during pregnancy affects cardiac health in offspring. When rats have marginally low copper intake during pregnancy and lactation, their adult, copper- repleted offspring exhibit increased heart mitochondrial hydrogen peroxide production and abnormally low activity of a copper-dependent enzyme, cytochrome c oxidase, that is important for cardiac mitochondrial energy production. Furthermore, the vascular relaxation response was altered in both the first and second generation offspring of the marginally copper deficient dams. These findings suggest that low copper intake by pregnant women may increase the risk for cardiovascular disease in their children and grandchildren. Mitochondrial dysfunction is a component in the development of several conditions such as heart disease, diabetes and obesity. Research into the role of sub-optimal intakes of protein, iron or zinc during pregnancy in promoting disease in offspring by adversely programming mitochondrial function during early development will continue in a new project titled �Mitochondrial Function and Nutritional Programming in the Prevention of Diet-Related Disease� (5450- 51000-041-00D).
Impacts
(N/A)
Publications
- Klevay, L.M., Aladjem, M., Bogden, J.D., Sandstead, H.H., Kemp, F.W., Li, W., Skurnick, J., Aviv, A. 2007. Renal and gastrointestinal potassium excretion in humans: new insight based on new data and review and analysis of published studies. Journal of the American College of Nutrition. 26(2) :103-110.
- Falcone, J.C., Lominadze, D., Johnson, W.T., Schuschke, D.A. 2008. Endothelial Cell-Derived Nitric Oxide Mobilization is Attenuated in Copper- Deficient Rats. Applied Physiology, Nutrition & Metabolism. 33:1073-1078.
- Nielsen, F.H. 2009. Marginal Zinc Deficiency Increases Magnesium Retention and Impairs Calcium Utilization in Rats. Biological Trace Element Research. 128(3):220-231.
- Zhou, Z., Johnson, W.T., Kang, Y.J. 2009. Regression of Copper-Deficient Heart Hypertrophy: Reduction in the Size of Hypertrophic Cardiomyocytes. Journal of Nutritional Biochemistry. 20(8):621-628.
- Johnson, W.T., Johnson, L.K. 2008. Copper Deficiency Inhibits CA2+-Induced Swelling in rat Cardiac Mitochondria. Journal of Nutritional Biochemistry. doi.10.1016/j.jnutbio.2008.02.009
- Johnson, W.T., Anderson, C.M. 2008. Cardiac Cytochrome c Oxidase Activity and Contents of Submits 1 and 4 are Altered in Offspring by Low Prenatal Intake by Rat Dams. Journal of Nutrition. 138:1269-1273
- Kang, Y.J., Jiang, Y., Saari, J.T. 2007. Changes in copper and zinc status and response to dietary copper deficiency in metallothionein- overexpressing transgenic mouse heart. Journal of Nutritional Biochemistry. 18(11):714-718.
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Progress 10/01/06 to 09/30/07
Outputs
Progress Report Objectives (from AD-416) Overall, to determine, using animal models, whether copper (Cu) intakes consistent with those observed in humans can adequately support cardiovascular functions. To develop a strategy for assessment of marginal copper deficiency in animals; to use this strategy to determine biomarkers of copper status that are suitable for assessment of marginal status in humans. To determine the contribution of oxygen- and nitrogen- derived reactive species to the cardiomyopathy (metabolic, contractile) induced by Cu deficiency, and the dietary intakes at which this pathology occurs. To determine whether low Cu intakes consistent with those observed in humans can impair nitric oxide-dependent control of blood vessels and blood pressure regulation. To determine whether the oxidative stress induced by Cu deficiency affects homocysteine metabolism and, thereby, cardiovascular function, and whether such effects influence nitric oxide-dependent signal transduction and/or other mechanisms that affect atherosclerosis. To determine whether marginal Zn deficiency can exacerbate or unmask cardiovascular effects of sub-optimal Cu status by virtue of its role in oxidative/nitrosative metabolism. Approach (from AD-416) Laboratory animals of varying ages and, in some cases, varying genetic makeup will be fed diets containing copper in severely deficient, marginally deficient or adequate amounts for varying periods of time. Based on comparisons to reliable invasive markers of copper status, non- invasive biomarkers will be tested for validity in assessing marginal copper deficiency. Tests of cardiovascular function and examinations of mechanism of depressed function will be made over ranges of copper status varying from severely-deficient to adequate. Cardiovascular functional measurements include heart contractile function, cardiac mitochondrial respiration and respiratory complex activity, blood vessel contractility and blood pressure. Atherosclerosis will be assessed by microscopic observation of blood vessels. Examination of mechanisms of depressed function will focus on the effects of oxidative stress and altered nitric oxide metabolism known to occur in copper deficiency. Such examination will include assessment of oxidative/nitrosative damage, altered nitric oxide signaling and altered homocysteine metabolism and will extend to measurement of transcription factors, mRNA and enzymes that influence and are influenced by oxidative stress and nitric oxide signaling. Accomplishments Copper supplementation reverses cardiac enlargement caused by pressure overload in mice. In experimental rodents, copper deficiency causes cardiac enlargement that can be reversed by restoring normal copper status. However, little is known about the influence of dietary copper on cardiac enlargement caused by cardiovascular diseases that increase pressure overload. In mice, surgical constriction of the aorta produces chronic pressure overload and eventual cardiac enlargement and failure. It was found that supplementing the mice with about 3 times the normal dietary Cu requirement reversed cardiac enlargement and prevented heart failure in the mice subjected to chronic pressure overload. Copper supplementation improved cardiac enlargement in part by promoting the biogenesis of blood vessels in the heart. IMPACT: Cardiac enlargement and heart failure are a major cause of disability and death in humans. This study suggests that dietary copper requirements may increase in people with certain types of heart disease associated with cardiac enlargement and that supplemental copper may be beneficial for improving cardiac function in these people. [NP 107 Action Plan Component 4: Nutrient Requirements; ARS Strategic Plan Performance Measures 4.1.2: Define functions, bioavailability, interactions, and human requirements (including effects such as genetic, health status, and environmental factors) for known, emerging, and new classes of nutrients in the food supply and provide that information in databases.] Low copper intake during pregnancy but not during lactation results in the reduction of cytochrome c oxidase subunits encoded by both mitochondrial and nuclear DNA in cardiac mitochondria of the offspring. One of our previous studies showed that reduced cardiac cytochrome c oxidase activity produced in the offspring of moderately copper-deficient rats resulted not from low copper intake during lactation, but from low intakes during pregnancy. The present study showed that the reduction in cytochrome oxidase activity resulting from low copper intake during pregnancy was accompanied by reductions in both the mitochondrial- and nuclear-encoded subunits of cytochrome c oxidase in cardiac mitochondria. Furthermore, the reduction in subunit content was not reversed by cross fostering the pups of copper-deficient dams to copper-adequate dams. This indicates that low copper intakes during pregnancy alters the programming of cardiac cytochrome c oxidase expression during fetal development. IMPACT: Dietary surveys indicate that pregnant women often do not meet the current recommended daily intake for dietary Cu. This finding indicates that moderately low Cu intake by pregnant women may alter the programmed development of the fetal heart in a manner that increases the risk of heart disease in their children because of impaired mitochondrial function. [NP 107 Action Plan Component 4: Nutrient Requirements; ARS Strategic Plan Performance Measures 4.1.2: Define functions, bioavailability, interactions, and human requirements (including effects such as genetic, health status, and environmental factors) for known, emerging, and new classes of nutrients in the food supply and provide that information in databases.] Technology Transfer Number of Non-Peer Reviewed Presentations and Proceedings: 5 Number of Newspaper Articles,Presentations for NonScience Audiences: 1
Impacts
(N/A)
Publications
- Jiang, Y., Reynolds, C., Xiao, C., Feng, W., Zhou, Z., Rodriguez, W., Tyagi, S., Eaton, J.W., Saari, J.T., Kang, Y.J. 2007. Dietary copper supplementation reverses hypertrophic cardiomyopathy induced by chronic pressure overload in mice. Journal of Experimental Medicine. 204(3):657- 666.
- Nielsen, F.H., Milne, D.B., Klevay, L.M., Gallagher, S., Johnson, L.K. 2007. Dietary magnesium deficiency induces heart rhythm changes, impairs glucose tolerance, and decreases serum cholesterol in post menopausal women. Journal of the American College of Nutrition. 26(2):121-132.
- Relling, D.P., Esberg, L.B., Johnson, W.T., Murphy, E.J., Carlson, E.C., Lukaski, H.C., Saari, J.T., Ren, J. 2007. Dietary interaction of high fat and marginal copper deficiency on cardiac contractile function. Obesity. 15(5):1242-1257.
- Saari, J.T., Reeves, P.G., Johnson, W.T., Johnson, L.K. 2006. Pinto beans are a source of highly bioavailable copper. Journal of Nutrition. 136:2999- 3004.
- Saari, J.T., Wold, L.E., Duan, J., Ren, J., Carlson, H.L., Bode, A.M., Lentsch, A.B., Zeng, H., Schuschke, D.A. 2006. Cardiac nitric oxide synthases are elevated in dietary copper deficiency. Journal of Nutritional Biochemistry. doi:10.1016/j.jnutbio.2006.07.006.
- Uthus, E.O., Reeves, P.G., Saari, J.T. 2007. Copper deficiency decreases plasma homocysteine in rats. Journal of Nutrition. 137:1370-1374.
- Zeng, H., Saari, J.T., Johnson, W.T. 2007. Copper deficiency decreases complex IV but not complex I, II, III, or V in the mitochondrial respiratory chain in rat heart. Journal of Nutrition. 137:14-18.
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Progress 10/01/05 to 09/30/06
Outputs
Progress Report 1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter? Cardiovascular disease is the leading cause of death in this country, with the direct annual cost projected to approach $250 billion in 2005. Basic research and epidemiological studies have indicated that inadequate dietary intakes of mineral elements such as calcium, copper, magnesium, zinc, as well as overload of iron are associated with altered functions of the heart and circulation. Furthermore, dietary surveys indicate that appreciable numbers of people have sub-optimal intakes of at least some of these minerals. However, we presently have little definitive proof that changing dietary practices with regard to these minerals will benefit cardiovascular health. A clear understanding of how these minerals, particularly at marginal intakes, contribute to cardiovascular function will
provide the basis for dietary recommendations that improve the health of the general public. The current project will focus on the contribution of dietary copper to cardiovascular health. The overall objective is to determine, using animal models, whether copper (Cu)intakes consistent with those observed in humans can adequately support cardiovascular functions. This objective will be pursued through the mechanistic tether of oxidative stress/altered nitric oxide metabolism by which Cu functions, and includes the following specific objectives: 1) to develop a strategy for assessment of marginal copper deficiency in animals; to use this strategy to determine biomarkers of copper status that are suitable for assessment of marginal status in humans, 2) to determine the contribution of oxygen- and nitrogen-derived reactive species to the cardiomyopathy (metabolic, contractile) induced by Cu deficiency, and the dietary intakes at which this pathology occurs, 3) to determine whether low Cu
intakes consistent with those observed in humans can impair nitric oxide-dependent control of blood vessels and blood pressure regulation, 4) to determine whether the oxidative stress induced by Cu deficiency affects homocysteine metabolism and, thereby, cardiovascular function, and whether such effects influence nitric oxide-dependent signal transduction and/or other mechanisms that affect atherosclerosis, 5) to determine whether marginal Zn deficiency can exacerbate or unmask cardiovascular effects of sub- optimal Cu status by virtue of its role in oxidative/nitrosative metabolism. Research will address components of National Program 107, Human Nutrition (100%). From component 1, Nutrition Requirements, objectives A (Biomarkers), B (Mechanism of Action), C (Nutrient Interactions), E (Genetic Variability) and G (Function and Performance) will be addressed. From component 2, Diet, Genetics, Lifestyle, and the Prevention of Obesity and Disease, objective A (Identify nutritional,
environmental and genetic factors that modify the effects of nutrient intake and metabolism on health outcomes) will be addressed. 2. List by year the currently approved milestones (indicators of research progress) Year 1 (FY 2005) Assess relationships between organ copper and marginal intakes of dietary copper; select organ with best discrimination of copper intake. Determine if copper deficiency causes reduced mitochondrial respiratory complex activity. Identify mitochondrial respiratory complexes causing increased hydrogen peroxide production during copper deficiency. Determine nitric oxide effect on mitochondrial respiration in copper deficiency. Determine effect of copper deficiency on homocysteine metabolism. Year 2 (FY 2006) Determine signaling pathway for induction of inducible nitric oxide synthase during copper deficiency. Determine nature of effect of altered nitric oxide on blood pressure during copper deficiency. Determine effect of copper deficiency on bilirubin and
biliverdin reductase. Determine if low zinc acts to exaggerate cardiovascular effects of marginal copper. Year 3 (FY 2007) Correlate organ copper content with semi-direct indicators of copper status; select best potential single biomarker(s). Identify respiratory complexes affected by developmental copper deficiency. Determine extent of oxidative modification of mitochondrial DNA by copper deficiency. Determine role of nitric oxide in impaired contractile function of copper deficiency. Determine relationship between nitric oxide, oxidative stress, homocysteine in atherosclerotic symptoms of copper deficiency. Clarify role of oxidative/nitrosative stress in zinc/copper interaction. Year 4 (FY 2008) Determine nature of effect of altered nitric oxide on coronary vessels during copper deficiency. Determine role of heme oxygenase in atherosclerotic effects of copper deficiency. Determine the ability of a diet high in antioxidants to ameliorate oxidant stress induced by low copper/low zinc.
[This is a new milestone (positive contingency) that anticipates the finding of a role for oxidative/nitrosative stress in the detrimental interaction between zinc and copper.] Year 5 (FY 2009) Correlate organ copper content with combinations of indicators of copper status; select best combination of indicators as biomarker. Identify oxidized, nitrated mitochondrial proteins in copper deficiency. Identify mitochondrial DNA mutations of copper deficiency. Relate mitochondrial DNA mutations to expression of respiratory complexes caused by copper deficiency. Determine whether elevation of inducible nitric oxide synthase preconditions copper-deficient hearts [contingency]. 4a List the single most significant research accomplishment during FY 2006. Reduced cardiac respiratory enzyme activity in the offspring of moderately Cu-deficient rats results from low dietary Cu during pregnancy, not during lactation: Our previous studies showed that low Cu intake by rats during pregnancy and
lactation reduced cytochrome c oxidase activity in cardiac mitochondria of the offspring. The reduction in enzyme activity was first observed late in the lactation phase of heart development and persisted in the offspring even after nine months of repletion with adequate dietary Cu. To determine whether this defect originated before or after birth, pups from moderately Cu-deficient dams were nursed by Cu-adequate dams and pups from Cu-adequate dams were nursed by moderately Cu- deficient dams from one day after birth. This cross-fostering did not reverse the reduction of heart cytochrome c oxidase activity in pups from Cu-deficient dams nor did it cause a defect in those from Cu-adequate dams. This indicates that the long-term loss of cytochrome c oxidase activity in the offspring of moderately Cu-deficient dams is caused by a defect in heart development before birth. IMPACT: Dietary surveys indicate that pregnant women often do not meet the current recommended daily intake for
dietary Cu. Our finding suggests that moderately low Cu intake by pregnant women could cause a developmental defect in the fetal heart that may increase the risk of heart disease in their children, particularly as the children become older, because of long-term impairment of mitochondrial function. [NP 107 Action Plan Component 4: Nutrient Requirements; ARS Strategic Plan Performance Measures 4.1.2: Define functions, bioavailability, interactions, and human requirements (including effects such as genetic, health status, and environmental factors) for known, emerging, and new classes of nutrients in the food supply and provide that information in databases.] 4b List other significant research accomplishment(s), if any. Copper deficiency lowers plasma homocysteine and affects mRNA expression of several enzymes involved in homocysteine/methionine metabolism: Homocysteine was found to be decreased in copper deficient rats. Homocysteine, a risk factor in cardiovascular disease, can be
metabolized (removed) in one of two ways. It can enter the irreversible transsulfuration pathway or it can be remethylated to form methionine. Our data suggest the remethylation of homocysteine by methionine synthase is increased in copper deficient rats. Further, mRNA for methylenetetrahydrofolate reductase, an enzyme that makes one of the substrates for methionine synthase, is also increased in copper deficiency. IMPACT: These results are contrary to published results and suggest that the risk of heart disease by copper deficiency is not mediated by elevation of homocysteine. [NP 107 Action Plan Component 4: Nutrient Requirements; ARS Strategic Plan Performance Measures 4.1.2: Define functions, bioavailability, interactions, and human requirements (including effects such as genetic, health status, and environmental factors) for known, emerging, and new classes of nutrients in the food supply and provide that information in databases.] Dietary copper deficiency selectively reduces
genetic expression of only one of five respiratory enzyme complexes in the heart: Dietary copper (Cu) deficiency is known to impair mitochondrial respiratory function, which is catalyzed by five membrane-bound multiple protein complexes. However, few studies have simultaneously analyzed the effect of Cu deficiency on the subunit protein expression of all five protein complexes. Examination of expression of subunits in each of the five respiratory complexes in Cu-deficient rat hearts revealed that only in the cytochrome c oxidase complex was the subunit expression reduced. IMPACT: This study further characterizes the defect in the ability of the heart to use oxygen under Cu-deficient conditions and thus contributes to our knowledge of heart failure in dietary Cu deficiency. [NP 107 Action Plan Component 4: Nutrient Requirements; ARS Strategic Plan Performance Measures 4.1.2: Define functions, bioavailability, interactions, and human requirements (including effects such as genetic,
health status, and environmental factors) for known, emerging, and new classes of nutrients in the food supply and provide that information in databases.] Dietary Cu deficiency increases nitric oxide production in rat hearts: Nitric oxide is a signaling molecule that, depending on how it is produced, can be either harmful or beneficial to heart function. We found that two enzymes that produce nitric oxide, one that initiates a pathway that leads to cell death and the other that leads to cell protection, are both elevated in dietary copper deficiency in rats. IMPACT: These findings contribute to the view that dietary copper deficiency promotes both heart failure and actions that compensate for this failure and suggest signaling mechanisms by which this may take place. [NP 107 Action Plan Component 4: Nutrient Requirements; ARS Strategic Plan Performance Measures 4.1.2: Define functions, bioavailability, interactions, and human requirements (including effects such as genetic, health
status, and environmental factors) for known, emerging, and new classes of nutrients in the food supply and provide that information in databases.] 5. Describe the major accomplishments to date and their predicted or actual impact. Long-term marginal copper deficiency in adult animals causes heart and blood vessel pathology: Dietary surveys indicate that many humans consume less than the recommended amount of copper. Although studies in animals indicate that dietary copper deficiency causes defects in cardiovascular (heart and blood vessel) structure and function, copper intake in these studies is generally too low to be relevant to human consumption. In a collaborative study with the University of Louisville, scientists at the GFHNRC found that if adult rats are fed marginally-deficient levels of copper, but for sufficiently long periods of time, they too exhibit defects in cardiovascular function and structure that are similar to those observed in severely-deficient young rats.
IMPACT: These findings, reported in two papers (Li et al, 2005; Falcone et al, 2005), illustrate that low copper intakes in animals that are comparable to those observed in humans can impair heart and blood vessel function. This provides a rationale for testing for such a possibility in humans. [NP 107 Action Plan Component 4: Nutrient Requirements; ARS Strategic Plan Performance Measures 4.1.2: Define functions, bioavailability, interactions, and human requirements (including effects such as genetic, health status, and environmental factors) for known, emerging, and new classes of nutrients in the food supply and provide that information in databases.] Pinto beans provide a bio-available source of dietary copper: Dry edible beans have been shown to be beneficial to cardiovascular health, but the active ingredient(s) for this benefit is(are) unknown. Because copper is known to be essential for cardiovascular health and beans are known to be a good source of copper, we hypothesize that
copper from beans could support cardiovascular health. In order to determine this, we first tested whether the copper from dry edible beans was bio- available. We found that when copper was fed to copper-deficient rats in the form of pinto beans, it was equally as effective as inorganic copper in restoring copper status indices (e.g., organ copper, activity of copper-dependent enzymes) to those rats. IMPACT: These findings indicate that the beans structure does not impede the delivery of its copper to the animal, which paves the way for testing of effectiveness of bean copper on cardiovascular function in both animals and humans. [NP 107 Action Plan Component 4: Nutrient Requirements; ARS Strategic Plan Performance Measures 4.1.2: Define functions, bioavailability, interactions, and human requirements (including effects such as genetic, health status, and environmental factors) for known, emerging, and new classes of nutrients in the food supply and provide that information in
databases.] Copper deficiency causes elevation of a structural elastic protein in the heart: We have demonstrated for the first time an 85% increase in fibulin-5 (also known as DANCE/EVEC) and a 71% decrease in cytochrome C oxidase (CCO) VIb subunit, but no change in succinate dehydrogenase complex (also known as complex II) IP subunit in Cu-deficient rat heart when compared with that of Cu-adequate rats. IMPACT: The elevation of fibulin-5, in particular, is important, because it implies that survival mechanisms have been initiated, which indirectly confirms that dietary copper deficiency leads to heart failure. [NP 107 Action Plan Component 4: Nutrient Requirements; ARS Strategic Plan Performance Measures 4.1.2: Define functions, bioavailability, interactions, and human requirements (including effects such as genetic, health status, and environmental factors) for known, emerging, and new classes of nutrients in the food supply and provide that information in databases.] 6. What
science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? The usual routine transfer of nutritional knowledge about the nutritional, beneficial, and non-beneficial effects of trace elements was made through direct contact with industry representatives and the public and with other scientists through presentations at national and international meetings and professional publications. 7. List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). Jack T. Saari wrote an article which appeared in 2006 in the nutrition section of the Grand Forks Herald titled Add Color to Your Diet with Fruits, Veggies. W. Thomas Johnson wrote an article which
appeared in September 2006 in the nutrition section of the Grand Forks Herald titled DASH Away High Blood Pressure. W. Thomas Johnson presented a talk in February 2006 at the Department of Anatomy and Cell Biology, University of North Dakota School of Medicine and Health Sciences titled Maternal Copper Deficiency Has Negative Cardiac Effects in the First Generation. W. Thomas Johnson presented a talk in August 2006 at the East Grand Forks Senior Citizen Center titled DASH Away High Blood Pressure.
Impacts
(N/A)
Publications
- Johnson, W.T., Brown-Borg, H.M. 2006. Cardiac cytochrome-c oxidase deficiency occurs during late postnatal development in progeny of copper- deficient rats. Experimental Biology and Medicine. 231:172-180.
- Wang, J., Song, Y., Elsherif, L., Song, Z., Zhou, G., Prabhu, S.D., Saari, J.T., Cai, L. 2006. Cardiac metallothionein induction plays the major role in the prevention of diabetic cardiomyopathy by zinc supplementation. Circulation. 113:544-554.
- Relling, D.P., Esberg, L.B., Fang, C.X., Johnson, W.T., Murphy, E.J., Carlson, E.C., Saari, J.T., Ren, J. 2006. High-fat diet-induced juvenile obesity leads to cardiomyocyte dysfunction and upregulation of Foxo3a transcription factor independent of lipotoxicity and apoptosis. Journal of Hypertension. 24:549-561.
- Johnson, W.T., Newman, Jr., S.M. 2006. Cardiac mitochondrial function is altered in the adult offspring of copper-deficient dams [abstract]. Journal of Federation of American Societies for Experimental Biology. 20(5) :A1065.
- Schuschke, D.A., Williams, C., Kang, Y.J., Saari, J.T. 2006. Cu-repletion promotes angiogenesis in the Cu-deficient rat heart [abstract]. Journal of Federation of American Societies for Experimental Biology. 20(4):A553.
- Saari, J.T., Reeves, P.G. 2006. Pinto beans are a good source of dietary copper [abstract]. Journal of Federation of American Societies for Experimental Biology. 20(4):A554.
- Zeng, H., Saari, J.T. 2006. New findings on protein expression on copper deficient rat heart with proteomic approach [abstract]. FASEB J. 20(4) :A553.
- Zeng, H., Saari, J.T., Dahlen, G.M. 2005. Copper deficiency increases fibulin-5 (dance/evec) but decreases cytochrome c oxidase vib expression in rat heart. Inorganic Biochemistry. 100:186-91.
- Reeves, P.G., Saari, J.T. 2005. Bioavailability of copper from cooked dry beans [abstract]. Annals of Nutrition and Metabolism. 49(Suppl 1). p.2.3.
- Combs, G.F. 2006. Indications of magnesium and calcium deficiency in populations [abstract]. International Symposium on Health Aspects of Calcium and Magnesium in Drinking Water, Program and Abstracts, p. 41.
- Nielsen, F.H. 2006. Arsenic. In: Klasing, K.C., editor. Mineral Tolerance of Animals. 2nd Revised Edition. Washington DC; National Academies Press. p. 31-41.
- Johnson, W.T., Newman Jr, S.M. 2007. Hearts in adult offspring of copper- deficient dams exhibit decreased cytochrome c oxidase activity, increased mitochondrial hydrogen peroxide generation and enhanced formation of intracellular residual bodies. Journal of Nutritional Biochemistry. 18:97- 104.
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Progress 10/01/04 to 09/30/05
Outputs
1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? Cardiovascular disease is the leading cause of death in this country, with the direct annual cost projected to approach $250 billion in 2005. Basic research and epidemiological studies have indicated that inadequate dietary intakes of mineral elements such as calcium, copper, magnesium, zinc, as well as overload of iron are associated with altered functions of the heart and circulation. Furthermore, dietary surveys indicate that appreciable numbers of people have sub-optimal intakes of at least some of these minerals. However, we presently have little definitive proof that changing dietary practices with regard to these minerals will benefit cardiovascular health. A clear understanding of how these minerals, particularly at marginal intakes, contribute to cardiovascular function will provide the basis
for dietary recommendations that improve the health of the general public. The current project will focus on the contribution of dietary copper to cardiovascular health. The overall objective is to determine, using animal models, whether copper (Cu) intakes consistent with those observed in humans can adequately support cardiovascular functions. This objective will be pursued through the mechanistic tether of oxidative stress/altered nitric oxide metabolism by which Cu functions, and includes the following specific objectives 1) to develop a strategy for assessment of marginal copper deficiency in animals; to use this strategy to determine biomarkers of copper status that are suitable for assessment of marginal status in humans, 2) to determine the contribution of oxygen- and nitrogen-derived reactive species to the cardiomyopathy (metabolic, contractile) induced by Cu deficiency, and the dietary intakes at which this pathology occurs, 3) to determine whether low Cu intakes consistent
with those observed in humans can impair nitric oxide-dependent control of blood vessels and blood pressure regulation, 4) to determine whether the oxidative stress induced by Cu deficiency affects homocysteine metabolism and, thereby, cardiovascular function, and whether such effects influence nitric oxide-dependent signal transduction and/or other mechanisms that affect atherosclerosis, 5) to determine whether marginal Zn deficiency can exacerbate or unmask cardiovascular effects of sub- optimal Cu status by virtue of its role in oxidative/nitrosative metabolism. Research will address components of National Program 107, Human Nutrition (100%). From Component 1, Nutrition Requirements, Objectives A (Biomarkers), B (Mechanism of Action), C (Nutrient Interactions), E (Genetic Variability) and G (Function and Performance) will be addressed. From Component 2, Diet, Genetics, Lifestyle, and the Prevention of Obesity and Disease, Objective A (Identify nutritional, environmental and genetic
factors that modify the effects of nutrient intake and metabolism on health outcomes) will be addressed. 2. List the milestones (indicators of progress) from your Project Plan. Year 1 (FY 2005) Assess relationships between organ copper and marginal intakes of dietary copper; select organ with best discrimination of copper intake. Determine if copper deficiency causes reduced mitochondrial respiratory complex activity. Identify mitochondrial respiratory complexes causing increased hydrogen peroxide production during copper deficiency. Determine nitric oxide effect on mitochondrial respiration in copper deficiency. Determine effect of copper deficiency on homocysteine metabolism. Year 2 (FY 2006) Determine signaling pathway for induction of inducible nitric oxide synthase during copper deficiency. Determine nature of effect of altered nitric oxide on blood pressure during copper deficiency. Determine effect of copper deficiency on bilirubin and biliverdin reductase. Determine if low
zinc acts to exaggerate cardiovascular effects of marginal copper. Year 3 (FY 2007) Correlate organ copper content with semi-direct indicators of copper status; select best potential single biomarker(s). Identify respiratory complexes affected by developmental copper deficiency. Determine extent of oxidative modification of mitochondrial DNA by copper deficiency. Determine role of nitric oxide in impaired contractile function of copper deficiency. Determine relationship between nitric oxide, oxidative stress, homocysteine in atherosclerotic symptoms of copper deficiency. Clarify role of oxidative/nitrosative stress in zinc/copper interaction. Year 4 (FY 2008) Determine nature of effect of altered nitric oxide on coronary vessels during copper deficiency. Determine role of hemoxygenase in atherosclerotic effects of copper deficiency. Year 5 (FY 2009) Correlate organ copper content with combinations of indicators of copper status; select best combination of indicators as biomarker.
Identify oxidized, nitrated mitochondrial proteins in copper deficiency. Identify mitochondrial DNA mutations of copper deficiency. Relate mitochondrial DNA mutations to expression of respiratory complexes caused by copper deficiency. [Determine whether elevation of inducible nitric oxide synthase preconditions copper-deficient hearts (contingency).] 3a List the milestones that were scheduled to be addressed in FY 2005. For each milestone, indicate the status: fully met, substantially met, or not met. If not met, why. 1. Assess relationships between organ copper and marginal intakes of dietary copper; select organ with best discrimination of copper intake. Milestone Substantially Met 2. Determine if copper deficiency causes reduced respiratory complex activity, and Identify respiratory complexes causing increased hydrogen peroxide. Milestone Substantially Met 3. Determine nitric oxide effect on mitochondrial respiration in copper deficiency. Milestone Not Met Progress slowed by
resource limitation (human,fiscal,equipment, etc. 4. Determine effect of copper deficiency on homocysteine metabolism. Milestone Not Met Other 5. Determine whether dry edible beans can supply copper to reverse indices of copper deficiency (pilot to test1). Milestone Substantially Met 3b List the milestones that you expect to address over the next 3 years (FY 2006, 2007, and 2008). What do you expect to accomplish, year by year, over the next 3 years under each milestone? Year 1 (FY 2006) Determine signaling pathway for induction of inducible nitric oxide synthase. Examine effects of copper deficiency on protein levels, mRNA, transcription and activation factors for isoforms of nitric oxide synthase in hearts of laboratory animals. The approach on this milestone will be altered somewhat by preceding the animal studies with cell culture techniques that use an immortalized mouse heart cell line and copper chelation techniques. We anticipate that this approach will greatly accelerate
progress toward identity of altered signalling pathways in copper-deficient hearts. IMPACT: These studies will aid in defining, on a molecular level, the basis for heart failure in dietary copper deficiency. Determine nature of effect of altered nitric oxide on blood pressure. Utilize pharmacological inhibition of nitric oxide synthase with L- arginine analogs, in combination with blockade of hormones responsible for blood pressure regulation, to determine the pathways involved in altered blood pressure regulation caused by dietary copper deficiency. IMPACT: Because dietary copper deficiency is known to impair blood vessel function by altered nitric oxide metabolism, this study will examine whether this impairment carries over to alteration of blood pressure. Determine effect of copper deficiency on bilirubin and biliverdin reductase. This milestone will not be pursued because of the retirement of the scientist proposing it. Determine if low zinc acts to exaggerate cardiovascular
effects of marginal copper. A factorially arranged experiment with rats will be performed to determine whether a moderate zinc deficiency is a stressor of copper metabolism or oxidative metabolism involving copper such that it increases the risk of cardiovascular dysfunction in marginal copper- deficiency. IMPACT: Because zinc and copper are known to affect each other's metabolism and because each is known to influence oxidative stress, this study will determine whether these two essential nutrients act synergistically in influencing cardiovascular function. Year 2 (FY 2007) Correlate organ copper content with semi-direct indicators of copper status; select best potential single biomarker(s). Utilizing long-term marginal copper deficiency studies in laboratory animals, correlate organ copper content with candidate indirect measures of copper status such as cytochrome c oxidase in platelets, extracellular superoxide dismutase in plasma, coagulation factors V and VIII, diamine oxidase,
copper chaperone for superoxide dismutase, to determine which ones are most sensitive to marginal copper intakes. IMPACT: Without sensitive, non-invasive indicators of copper status, the role of copper in human cardiovascular function is difficult to assess. These studies are aimed at providing candidate biomarkers of copper status for potential use in human studies. Identify respiratory complexes affected by developmental copper deficiency. Respiratory complex activities and protein subunits of complex I and complex IV will be assayed in the hearts of neonates from marginally Cu- deficient dams at various ages up to 1 year. IMPACT: Parallel reductions in complex activities and protein subunit content in cardiac mitochondria indicate that marginal Cu intakes during pregnancy leads to prolonged impairment of mitochondria function in the first generation and increases risk for developing age-related cardiac disease. Determine extent of oxidative modification of mitochondrial DNA by
copper deficiency. The content of 8-hydroxydeoxyguanosine will be measured in heart mitochondrial DNA from rats fed diets containing deficient, marginal, and adequate levels of copper. IMPACT: Increased 8-hydroxydeoxyguanosine indicates that mitochondrial DNA can be oxidatively modified during Cu deficiency. This can cause mutations in mitochondrial DNA that permanently impair mitochondrial function and lead to cardiac disease. Determine role of nitric oxide in heart contractile function. Determine by acute and chronic inhibition of nitric oxide synthase whether nitric oxide plays a role in altered contractile function of hearts from copper-deficient animals. IMPACT: This study is aimed at determining the mechanism of action of low dietary copper in impairing heart function. Determine relationship between nitric oxide, oxidative stress, homocysteine in atherosclerotic symptoms of copper deficiency. A series of experiments will be initiated that will be aimed at determining the
relative effects and interactions between nitric oxide, oxidative stress and homocysteine in causing atherosclerotic vessel damage in marginally copper-deficient animals. IMPACT: This study will examine the molecular mechanism of the now well-established impairment of blood vessel function caused by dietary copper deficiency. Clarify role of oxidative/nitrosative stress in zinc/copper interaction. If it is determined that a moderate zinc deficiency exacerbates signs of marginal copper deficiency, an experiment with rats will be performed to determine whether cardiovascular effects are modified by changing nitric oxide formation. IMPACT: If, in the year 2006 study, zinc proves to have an effect on copper-dependent alteration of cardiovascular function, a nitric oxide mechanism will be tested to further our knowledge of the molecular events affected by these two nutrients. Year 3 (FY 2008) Determine nature of effect of altered nitric oxide on coronary vessels during copper deficiency.
Hearts of animals fed varying levels of dietary copper will be isolated and examined for coronary blood vessel response to nitric oxide donors, selective nitric oxide synthase inhibitors, antioxidants and nitric oxide scavengers to assess whether copper deficiency causes pathologic dysfunction in coronary vessels via reactive oxygen or nitrogen species is it does in systemic blood vessels. IMPACT: The well known alteration of blood vessel function in the peripheral circulation will be examined directly in blood vessels of the heart. This will help to determine whether the known effect of copper deficiency on heart cells is caused by or exacerbated by an effect on the heart's circulation. Determine role of hemoxygenase in atherosclerotic effects of copper deficiency. Rats of varying copper status will be subjected to carotid artery damage and then treated with inhibitors of heme oxygenase (metalloprotoporphyrins) to determine the extent to which this enzyme plays a role in the
atherosclerotic effects of dietary copper deficiency. IMPACT: This study will further delineate the mechanism of action of low dietary copper in impaired blood vessel function. 4a What was the single most significant accomplishment this past year? Long-term marginal copper deficiency in adult animals causes heart and blood vessel pathology. Dietary surveys indicate that many humans consume less than the recommended amount of copper. Although studies in animals indicate that dietary copper deficiency causes defects in cardiovascular (heart and blood vessel) structure and function, copper intake in these studies is generally too low to be relevant to human consumption. In a collaborative study with the University of Louisville, scientists at the GFHNRC found that if adult rats are fed marginally-deficient levels of copper, but for sufficiently long periods of time, they too exhibit defects in cardiovascular function and structure that are similar to those observed in severely-deficient
young rats. IMPACT: These findings, reported in two papers (Falcone et al, 2005; another in press), illustrate that low copper intakes in animals that are comparable to those observed in humans can impair heart and blood vessel function. This provides a rationale for testing for such a possibility in humans. 4b List other significant accomplishments, if any. Pinto beans provide a bio-available source of dietary copper. Dry edible beans have been shown to be beneficial to cardiovascular health, but the active ingredient(s) for this benefit is(are) unknown. Because copper is known to be essential for cardiovascular health and beans are known to be a good source of copper, we hypothesize that copper from beans could support cardiovascular health. In order to determine this we first tested whether the copper from dry edible beans was bio- available. We found that when copper was fed to copper-deficient rats in the form of pinto beans, it was equally as effective as inorganic copper in
restoring copper status indices (e.g., organ copper, activity of copper-dependent enzymes) to those rats. IMPACT: These findings, being prepared for a meeting presentation, indicate that the bean's structure does not impede the delivery of its copper to the animal, which paves the way for testing of effectiveness of bean copper on cardiovascular function in both animals and humans. Copper deficiency causes elevation of a structural elastic protein in the heart. We have demonstrated for the first time that there is an 85% increase in fibulin-5 (also known DANCE/EVEC) and a 71% decrease in cytochrome C oxidase (CCO) VIb subunit, but no change in succinate dehydrogenase complex (also known complex II) IP subunit in Cu-deficient rat heart when compared with that of Cu-adequate rats. IMPACT: The elevation of fibulin- 5, in particular, is important, because it implies that survival mechanisms have been initiated, which indirectly confirms that dietary copper deficiency leads to heart
failure. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. [This report is the first for this CRIS project (certified in 2004), thus the accomplishments for the life of the project are incorporated in sections 4a and 4b. The following are related accomplishments that immediately preceded this CRIS project and thus provide background and continuity to the studies of the project.] Copper deficiency causes signs of heart failure. Although many contractile, electrical and metabolic abnormalities have been found in hearts of copper-deficient animals, characteristics that unequivocally indicate heart failure have not been demonstrated. Scientists at the Grand Forks Human Nutrition Research Center, in collaboration with scientists at the University of Louisville, measured variables in hearts of copper-deficient mice that are used in humans to demonstrate heart failure. Hearts of copper-deficient mice were found to have reduced
maximum contractile pressure, elevated pressure during relaxation, reduced responsiveness to excitation by adrenalin and increased collagen deposits, all of which are signs of heart failure. IMPACT: This suggests that dietary copper deficiency is a risk factor for heart disease and subsequent cardiac failure. NP 107 Action Plan Component 4: Nutrient Requirements; ARS Strategic Plan Performance Measures 4.1.2: Define functions, bioavailability, interactions, and human requirements (including effects such as genetic, health status, and environmental factors) for known, emerging, and new classes of nutrients in the food supply and provide that information in databases. Impaired blood pressure regulation in copper deficiency is related to nitric oxide. Although prior studies have shown that blood vessels of copper- deficient rats respond poorly to agents that stimulate release of nitric oxide, a potent endogenous dilator, it has not been shown that this causes an alteration of blood
pressure. In studies at the Grand Forks Human Nutrition Research Center, rats were injected with a drug (L-NAME) that inhibits formation of nitric oxide and elevates blood pressure. The elevation of blood pressure by L-NAME injection was found to be depressed in rats that were fed inadequate copper. IMPACT: This indicates that dietary copper deficiency causes an impairment of blood pressure regulation and a predisposition to high blood pressure. NP 107 Action Plan Component 4: Nutrient Requirements; ARS Strategic Plan Performance Measures 4.1.2: Define functions, bioavailability, interactions, and human requirements (including effects such as genetic, health status, and environmental factors) for known, emerging, and new classes of nutrients in the food supply and provide that information in databases. Copper deficiency enhances atherosclerosis-like blood vessel damage. Although dietary copper deficiency has been shown to enhance inflammation, the relationship between dietary copper
and blood vessel injury has not been previously examined. The Grand Forks Human Nutrition research Center, in collaboration with collaborators at the University of Louisville, examined effects of injury induced by balloon inflation within blood vessels of copper-deficient and copper-adequate rats. Blood vessel injury by balloon inflation within the vessel caused thickening of the blood vessel wall (akin to atherosclerosis) that was exaggerated by restriction of dietary copper. IMPACT: This finding provides further support for the view that the inflammatory response to injury is exaggerated by copper deficiency and, further, because balloon inflation of blood vessels (angioplasty) is a common mode of treatment for atherosclerotic blood vessels, the findings suggest that proper copper nutrition may improve the results of such treatment. NP 107 Action Plan Component 4: Nutrient Requirements; ARS Strategic Plan Performance Measures 4.1.2: Define functions, bioavailability, interactions,
and human requirements (including effects such as genetic, health status, and environmental factors) for known, emerging, and new classes of nutrients in the food supply and provide that information in databases. Copper deficiency during pregnancy causes long-term effects in offspring. Women of child-bearing age consume less than the recommended daily requirement of copper, but it is not known if low dietary copper intake during pregnancy has long-term effects on the cardiovascular system of children. To test this possibility in laboratory animals, scientists at the Grand Forks Human Nutrition Research Center bred rats to bear and nurse offspring during maternal copper deficiency; pups were then fed copper-adequate diets for nine months following weaning and assessed for altered heart mitochondrial function, enzymes and oxidative stress. Activities of respiratory complexes of heart mitochondria in neonates from copper deficient dams was significantly reduced at 21 days following birth
and could not be restored to normal activity levels by 6 weeks of copper supplementation. Further, despite nine months of copper repletion, rats of copper-deficient dams exhibited an abnormally low activity of cytochrome c oxidase, a copper-dependent enzyme, in heart mitochondria and an increase in heart mitochondrial hydrogen peroxide production. IMPACT: This finding indicates that copper deficiency during pregnancy has long-term, possibly irreversible, effects on energy metabolism in the hearts of neonates that could increase their susceptibility to heart disease as adults. NP 107 Action Plan Component 4: Nutrient Requirements; ARS Strategic Plan Performance Measures 4.1.2: Define functions, bioavailability, interactions, and human requirements (including effects such as genetic, health status, and environmental factors) for known, emerging, and new classes of nutrients in the food supply and provide that information in databases. 6. What science and/or technologies have been
transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? The usual routine transfer of nutritional knowledge about the nutritional, beneficial, and non-beneficial effects of trace elements was made through direct contact with industry representatives and the public and with other scientists through presentations at national and international meetings and professional publications. 7. List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). J.T. Saari wrote an article which appeared in January 2005 in the nutrition section of the Grand Forks Herald entitled "Being Heart Healthy Doesnt Need to be Hard." W.T. Johnson wrote an article which appeared in June 2005 in the nutrition
section of the Grand Forks Herald entitled "Copper A Building Block for a Healthy Baby."
Impacts
(N/A)
Publications
- Johnson, W.T., Newman, S.M., Jr. 2003. Copper deficiency: A potential model for determining the role of mitochondria in cardiac aging. Journal of the American Aging Association. 26:29-38.
- Klevay, L.M., Christopherson, D.M., Shuler, T.R. 2004. Hair as a biopsy material: trace element data on one man over two decades. European Journal of Clinical Nutrition. 58:1359-1364.
- Raymond, L.J., Johnson, W.T. 2004. Supplemental ascorbate or alpha- tocopherol induces cell death in Cu-deficient HL-60 cells. Experimental Biology and Medicine. 229:885-894.
- Zeng, H., Saari, J.T. 2004. Increased type I collagen content and DNA binding activity of a single-stranded, cytosine-rich sequence in the high- salt buffer protein extract of the copper-deficient rat heart. Journal of Nutritional Biochemistry. 15:694-99.
- Falcone, J.C., Saari, J.T., Kang, Y.J., Schuschke, D.A. 2005. Vasoreactivity in an adult rat model of marginal copper deficiency. Nutrition Research. 25:;177-186.
- Dong, F., Esberg, L.B., Roughead, Z.K., Ren, J., Saari, J.T. 2005. Increased contractility of cardiomyocytes from copper-deficient rats is associated with upregulation of cardiac insulin-like growth factor-I receptor. American Journal of Physiology Heart and Circulatory Physiology. 289:78-84.
- Song, Y., Wang, J., Li, Y., Du, Y., Arteel, G.E., Saari, J.T., Kang, Y.J., Cai, L. 2005. Cardiac metallothionein synthesis in streptozotocin-induced diabetic mice, and its protection against diabetes-induced cardiac injury. American Journal of Pathology. 167(1):17-26.
- Wang, L., Zhou, Z., Saari, J.T., Kang, Y.J. 2005. Alcohol-induced myocardial fibrosis in metallothionein-null mice: Prevention by zinc supplementation. American Journal of Pathology. 167(2):337-344.
- Zhou, A., Wang, L., Song, Z., Saari, J.T., McClain, C.J., Kang, Y.J. 2005. Zinc supplementation prevents alcoholic liver injury in mice through attenuation of oxidative stress. American Journal of Pathology. 166(6) :1681-1690.
- Klevay, L.M. 2005. Copper. In: Coates, P.M., Blackman, M.R., Cragg, G., Levine, M., Moss, J., White, J., editors. Encyclopedia of Dietary Supplements. New York: Marcel Dekker/Taylor and Francis Group. p. 133-141.
- Klevay, L.M., Combs, G.F. 2003. Mineral elements related to cardiovascular health. Proceedings of the World Health Organization Workshop, Rome, Italy, November 11-13, 2003.
- Saari, J.T., Dong, F., Esberg, L.B., Roughead, Z.K., Ren, J. 2005. Increased contractility is associated with increased cardiac IGF-I receptor protein in copper-deficient cardiomyocytes [abstract]. The Federation of American Societies for Experimental Biology Journal. 19(5) :A1486.
- Schuschke, D.A., Gordon, S.A., Saari, J.T., Lentsch, A.B. 2005. Impaired deformability of copper-deficient neutrophils [abstract]. The Federation of American Societies for Experimental Biology Journal. 19(5):A1485.
- Johnson, W.T. 2005. Copper and brain function. In: Lieberman, H.R., Kanarek, R.B., Prasad, C., editors. Nutritional Neuroscience. Boca Raton, FL:CRC Press/Taylor & Francis Group. p. 289-305.
- Johnson, W.T., Lukaski, H.C. 2005. Serum superoxide dismutase (SOD) activity is an index of copper status [abstract]. The Federation of American Societies for Experimental Biology Journal. 19(5):A1485.
- Li, Y., Wang, L., Schuschke, D.A., Zhou, Z., Saari, J.T. 2005. Marginal dietary copper restriction induces cardiomyopathy in rats. Journal of Nutrition. 135:2130-2136.
- Gordon, S.A., Lominadze, D., Saari, J.T., Lentsch, A.B., Schuschke, D.A. 2005. Impaired deformability of copper-deficient neutrophils. Experimental Biology and Medicine. 230:543-548.
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