Progress 05/27/04 to 04/30/09
Outputs
Progress Report Objectives (from AD-416) Obj. 1. Evaluate the role of nitric oxide in the outcome of coxsackievirus B3-induced acute myocarditis in selenium (Se) and/or vitamin E-deficient mice as a model of human inflammatory heart muscle disease. Obj. 2. Clarify the mechanism whereby dietary iron overload in the host leads to severe and unusual genomic mutations in coxsackievirus B3 inoculated into mice deficient in vitamin E. Obj. 3. Define biomarkers that differentiate between the proper elevated cancer chemopreventive dose of Se and the threshold toxic dose of Se through the application of proteomic discovery techniques. Approach (from AD-416) This project plan consists of three parts, each of which will examine the relationship between selenium and/or vitamin E status and human health. We will investigate the role of nitric oxide (NO) in the course of infection with the cardiotrophic coxsackievirus in mice as a model of human acute myocarditis. We hypothesize that greater heart muscle damage will occur in virus-infected and Se-deficient mice because viral infection and Se deficiency both increase NO in vivo. The second part will study the mechanism whereby dietary iron overload causes changes in the genome of coxsackievirus inoculated into vitamin E-deficient mice. This could serve as a model for various human diseases that are increased by iron excess including liver cancer and hepatitis. The third part will utilize proteomics in an attempt to characterize biomarkers that can distinguish between the elevated doses of selenium needed to demonstrate its cancer chemopreventive properties versus the dose that results in chronic selenium poisoning. If increased intakes of selenium are ever advocated as a cancer preventative public health measure, better ways of assessing selenium overexposure will be needed. Significant Activities that Support Special Target Populations The Lead scientist retired. This project responded to Nutrition Program 107, Action Plan 3.1.1, because it addressed studies of trace element nutrition to determine biochemical and health consequences of sub-optimal trace element intake, nutritional factors that determine how nutritional status and the immune system interact, evaluation of the role of diets in affecting risk factors for disease in humans, and nutritional requirements to determine the role of dietary antioxidants to control oxidative stress and the nutritional regulation of the immune response. No progress to report due to retirement. NPS approved the funds to be redirected to 1235-51000-046-00D, Bioavailability and Metabolism of Phytochemicals and Micronutrients, and 1235-51530-008-00D, Dietary Modulation of Markers of Inflammation and Oxidation as Risk Factors of Chronic, Degenerative Diseases.
Impacts
(N/A)
Publications
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Progress 10/01/06 to 09/30/07
Outputs
Progress Report Objectives (from AD-416) Obj. 1. Evaluate the role of nitric oxide in the outcome of coxsackievirus B3-induced acute myocarditis in selenium (Se) and/or vitamin E-deficient mice as a model of human inflammatory heart muscle disease. Obj. 2. Clarify the mechanism whereby dietary iron overload in the host leads to severe and unusual genomic mutations in coxsackievirus B3 inoculated into mice deficient in vitamin E. Obj. 3. Define biomarkers that differentiate between the proper elevated cancer chemopreventive dose of Se and the threshold toxic dose of Se through the application of proteomic discovery techniques. Approach (from AD-416) This project plan consists of three parts, each of which will examine the relationship between selenium and/or vitamin E status and human health. We will investigate the role of nitric oxide (NO) in the course of infection with the cardiotrophic coxsackievirus in mice as a model of human acute myocarditis. We hypothesize that greater heart muscle damage will occur in virus-infected and Se-deficient mice because viral infection and Se deficiency both increase NO in vivo. The second part will study the mechanism whereby dietary iron overload causes changes in the genome of coxsackievirus inoculated into vitamin E-deficient mice. This could serve as a model for various human diseases that are increased by iron excess including liver cancer and hepatitis. The third part will utilize proteomics in an attempt to characterize biomarkers that can distinguish between the elevated doses of selenium needed to demonstrate its cancer chemopreventive properties versus the dose that results in chronic selenium poisoning. If increased intakes of selenium are ever advocated as a cancer preventative public health measure, better ways of assessing selenium overexposure will be needed. Significant Activities that Support Special Target Populations This research supports National Program 107 Action Plan for Human Nutrition, Component 6, Prevention of Obesity and Disease: Relationship between Diet, Genetics, and Lifestyle because it addresses studies of trace element nutrition to determine biochemical and health consequence of sub�optimal trace element intake. Progress has been slowed during FY07 with the retirement of the lead scientist. Accomplishments Measurement of Nitric Oxide levels in Macrophages Activated Where Selenium is Deficient. Expression of selenium containing proteins appears to mark conversion of classically activated macrophages that respond to interferon gamma with the production of nitric oxide (NO) to alternatively activated macrophages that restrict NO production towards metabolism of arginine through arginase 1, an enzyme. This is important because NO is important for killing of bacteria and parasites by macrophages that engulf these bugs, while arginase 1 activity is important for wound healing but not for intracellular killing mechanisms. Measurement of levels of NO released by the action of inducible nitrix oxide synthase (iNOS) and arginase 1 activity demonstrate that this conversion occurs and that macrophage function is dependent on selenium. Initial studies detailed the expression of these activities in peritoneal macrophages activated by bacterial or parasite products to determine the changing patterns of the cells isolated from mice and after culture in vitro. Studies are in progress to evaluate the limitations of expression in mice made deficient in dietary selenium, and in cultures where the supporting media composition is depleted of selenium. These assays provide important tools for assessing macrophage function in response to infection and tissue repair, and the role of a dietary factor, selenium, in optimizing healthy outcomes. This project responds to National Program 107, Human Nutrition, Component3, Relationship Between Diet, Genetics, and Lifestyle and the Risk For Chronic Disease. Technology Transfer Number of Non-Peer Reviewed Presentations and Proceedings: 1
Impacts
(N/A)
Publications
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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? The project responds to Nutrition Program 107, Action Plan 3.1.1, because it addresses studies of trace element nutrition to determine biochemical and health consequences of sub-optimal trace element intake, nutritional factors that determine how nutritional status and the immune system interact, evaluation of the role of diets in affecting risk factors for disease in humans, and nutritional requirements to determine the role of dietary antioxidants to control oxidative stress and the nutritional regulation of the immune response. Cancer is responsible for 500,000 deaths annually in the United States alone, and inflammatory heart muscle disease kills 400,000 worldwide every year. Together both diseases result in economic losses of several billions of dollars per year on the general
population in the U.S. The focus of the research described in this project is to use animal models to determine whether or not the nutritional antioxidants selenium and vitamin E have any beneficial effect on the prevention of cancer or heart muscle inflammation. The experimental approach will involve the use of viral infection or dietary manipulation to determine the effect of such metabolic stressors on the ability of test animals (mice) to resist the development of disease. Improvement in disease outcome would serve as the basis for studies in humans to use dietary recommendations to reduce or limit the intensity of disease. 2. List by year the currently approved milestones (indicators of research progress) Milestone 1 (1 - 2 years) - The research will 1) develop general laboratory procedures to measure nitric oxide (NO) and conduct experiments to relate resistance to coxsackievirus B3 (CVB3) with NO production, 2) determine CVB3 genome changes in vitamin E (VE)-deficient mice
given iron (Fe) overload, and 3) prepare samples for SELDI-TOF analysis. Milestone 2 (2 - 3 years) - The research will 1) develop HPLC procedures for peroxynitrite and hydroxyl radical detection in liver and heart samples, 2) assay for plasma VE and Fe, and 3) initiate study of selenosis for adequate sample evaluation. Milestone 3 (3 - 4 years) - The research will 1) measure interaction between Se deficiency and CVB3 infection on NO production, 2) repeat study of VE deficiency and Fe overload diet on genome changes in CVB3, and 3) final evaluation of proteomic changes by SELDI-TOP. Milestone 4 (4 - 5 years) - The research will 1) determine sequence information on CVB3 genome from VE deficient, Fe overload study, 2) verify proteomic analysis using 2D gel electrophoresis, and 3) conduct pharmacological inhibition and use of genetic iNOS knockout mouse studies on CVB3 infected mice. 4a List the single most significant research accomplishment during FY 2006. Dietary restriction increases
the pathogenesis of coxsackievirus B3 infections: Restricted food intake is gaining greater acceptability by the general public as a life extension technique, but the long-term consequences of such a dietary anti-aging strategy is unknown. Studies with mice show that even a mild-moderate food withdrawal results in a highly undesirable side effect of viral infection (more viral pathogenesis). Thus, the use of food restriction as an approach to a prolonged lifespan may be too dangerous for routine use. 4b List other significant research accomplishment(s), if any. Increased oxidative stress in vivo leads to increased virulence of viral infection: The project responds to Nutrition Program 107, Action Plan 3.1.1, because it addresses studies of nutritional requirements that determine the role of dietary antioxidants to control oxidative stress and the nutritional regulation of the immune response. Copper deficiency exacerbates infection with the coxsackievirus, presumably by increasing
oxidative stress as superoxide dismutase activity is decreased. This is yet another example of how nutritional status can have a profound influence on the ability of a host to resist infection. Dietary selenium reverses congestive heart failure due to selenium deficiency: In collaboration with scientists form the University of Buenos Aires, it was shown that adding selenium to the diet of mice with heart disease caused by selenium deficiency improved contractile (inotropic) force and the frequency (chronotropic) of heart beats. Taken together, these results suggest that selenium may play a significant role in protecting against heart disease in those geographic areas where dietary selenium is too low. 4d Progress report. The project responds to Nutrition Program 107, Action Plan 3.1.1, because it addresses studies of trace element nutrition to determine biochemical and health consequences of sub-optimal trace element intake, nutritional factors that determine how nutritional status
and the immune system interact, evaluation of the role of diets in affecting risk factors for disease in humans, and nutritional requirements to determine the role of dietary antioxidants to control oxidative stress and the nutritional regulation of the immune response. Chinese scientists demonstrated that Se supplementation prevented Keshan disease, a cardiomyopathy that afflicts infants or young children who reside in areas with low Se. Collaborative studies with scientists at the University of Buenos Aires demonstrated that atrial tissue from mice fed a Se-deficient diet for 4 weeks postweaning had a diminished beta- adrenoceptor-inotropic cardiac response to isoproterenol or norepinephrine, compared with atrial tissue from mice fed the same diet supplemented with 0.2 ppm Se. The dose response curves of isoproterenol and norepinephrine were shifted to the right in Se-deficient mice and the potency of the beta-adrenoceptor agonist was diminished. This diminished response could be
reversed by pre-treating mice with nitric oxide synthase inhibitors such as N-monomethyl-L-arginine or aminopyridine. Elevated serum concentrations of nitrite/nitrate as well as a 3-fold increase in the heart muscle NOS activity was seen in the Se- versus the Se+ mice. Western blotting as well as indirect immunofluorescence indicated an enhanced expression of inducible nitric oxide synthase in hearts of Se- mice. Increased expression and activity of nitric oxide synthase, and increased nitrite/nitrate levels in Se- mice correlated with an impaired response to beta-adrenergic-isotropic cardiac stimulation. Thus, elevated nitric oxide levels due to Se deficiency may account for some of the cardiopathophysiology observed in these nutritionally-deprived animals. Others have noted the multiple physiological and pathophysiological effects of nitric oxide in cardiac function under conditions of nutritional adequacy. In summary, the following experimental observations have been made in the
mouse model: 1) Se deficiency leads to increased cardiac production of nitric oxide; 2) CVB3 infection leads to increased cardiac production of nitric oxide; 3) Se deficiency leads to increased cardiac damage due to coxsackievirus B3 infection. Therefore, the major goal of the experiments outlined in the project statement is to test the hypothesis that localized increased nitric oxide production in the heart in response to infection with coxsackievirus B3 induces heart muscle damage seen in Se-deficient mice. These studies will have impact on clinical observations of cardiopathology where elements of diet are a factor to be considered. 5. Describe the major accomplishments to date and their predicted or actual impact. The project responds to Nutrition Program 107, Action Plan 3.1.1, because it addresses studies of trace element nutrition to determine biochemical and health consequences of sub-optimal trace element intake, nutritional factors that determine how nutritional status and
the immune system interact, evaluation of the role of diets in affecting risk factors for disease in humans, and nutritional requirements to determine the role of dietary antioxidants to control oxidative stress and the nutritional regulation of the immune response. Chinese scientists demonstrated that Se supplementation prevented Keshan disease, a cardiomyopathy that afflicts infants or young children who reside in areas with low Se. Collaborative studies with scientists at the University of Buenos Aires demonstrated that atrial tissue from mice fed a Se-deficient diet for 4 weeks postweaning had a diminished beta- adrenoceptor-inotropic cardiac response to isoproterenol or norepinephrine, compared with atrial tissue from mice fed the same diet supplemented with 0.2 ppm Se. The dose response curves of isoproterenol and norepinephrine were shifted to the right in Se-deficient mice and the potency of the beta-adrenoceptor agonist was diminished. This diminished response could be
reversed by pre-treating mice with nitric oxide synthase inhibitors such as N-monomethyl-L-arginine or aminopyridine. Elevated serum concentrations of nitrite/nitrate as well as a 3-fold increase in the heart muscle NOS activity was seen in the Se- versus the Se+ mice. Western blotting as well as indirect immunofluorescence indicated an enhanced expression of inducible nitric oxide synthase in hearts of Se- mice. Increased expression and activity of nitric oxide synthase, and increased nitrite/nitrate levels in Se- mice correlated with an impaired response to beta-adrenergic-isotropic cardiac stimulation. Thus, elevated nitric oxide levels due to Se deficiency may account for some of the cardiopathophysiology observed in these nutritionally-deprived animals. Others have noted the multiple physiological and pathophysiological effects of nitric oxide in cardiac function under conditions of nutritional adequacy. In summary, the following experimental observations have been made in the
mouse model: 1) Se deficiency leads to increased cardiac production of nitric oxide; 2) CVB3 infection leads to increased cardiac production of nitric oxide; 3) Se deficiency leads to increased cardiac damage due to coxsackievirus B3 infection. Therefore, the major goal of the experiments outlined in the project statement is to test the hypothesis that localized increased nitric oxide production in the heart in response to infection with coxsackievirus B3 induces heart muscle damage seen in Se-deficient mice. These studies will have impact on clinical observations of cardiopathology where elements of diet are a factor to be considered. 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? Development of a proteomic analysis of serum and tissues from
animals with various levels of Se status will provide a tool that could be used to screen for dietary changes in Se that would be indicative of the appropriateness of Se status to control of viral infection. These markers could be of additional benefit to other diseases in which Se functions to improve health status such as has been suggested for cancer. This technology, when tested in humans, would be of benefit as a useable marker of Se status.
Impacts
(N/A)
Publications
- South, P.K., Smith, A.D., Guidry, C.A., Levander, O.A. 2006. Effect of physical restraint on oxidative stress in mice fed a selenium and vitamin E deficient diet. Biological Trace Element Research. 109(3):293-300.
- Levander, O.A., Burk, R.F. 2006. Update of human dietary standards for selenium. In: Hatfield, D.L., Berry, M.J., Gladyshev, V.N., editors. Selenium: Its Molecular Biology and Role in Human Health. 2nd edition. New York, NY: Springer Science and Business Media. p.399-410.
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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? Cancer is responsible for 500,000 deaths annually in the United States alone and inflammatory heart muscle disease kills 400,000 worldwide every year. Together both diseases result in economic losses of several billions of dollars per year on the general population in the U.S. The focus of the research described in this project is to use animal models to determine whether or not the nutritional antioxidants selenium (Se) and vitamin E (VE) have any beneficial effect on the prevention of cancer or heart muscle inflammation. The experimental approach will involve the use of viral infection or dietary manipulation to determine the effect of such metabolic stressors on the ability of test animals (mice) to resist the development of disease. Improvement in disease outcome would serve as the basis for
studies in humans to use dietary recommendations to reduce or limit the intensity of disease. The project responds to National Program 107 Action Plan 3.1.1 because it addresses studies of trace element nutrition to determine biochemical and health consequences of sub optimal trace element intake, nutritional factors that determine how nutritional status and the immune system interact, evaluation of the role of diets in affecting risk factors for disease in humans, and nutritional requirements to determine the role of dietary antioxidants to control oxidative stress and the nutritional regulation of the immune response. 2. List the milestones (indicators of progress) from your Project Plan. Milestone 1 (12 - 24 months) - Evaluate the role of nitric oxide (NO)in the outcome of coxsackievirus B3 (CBV3)-induced acute myocarditis in Se and/or VE-deficient mice as a model of human inflammatory heart muscle disease. Milestone 2 (24 - 48 months) - Clarify the mechanism whereby dietary iron
overload in the host leads to severe and unusual genomic mutations in coxsackievirus B3 inoculated into mice deficient in VE. Milestone 3 (36 - 48 months) - Define biomarkers that differentiate precisely between the proper elevated cancer chemopreventive dose of Se and the threshold toxic dose of Se through the application of proteomic discovery techniques. Milestone 4 (48 - 60 months) Complete technology transfer through the recommendation of appropriate levels of Se and VE that can be safely used to provide adequate antioxidant levels to respond to infectious agents without exacerbated pathology. 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. Evaluate the role of nitric oxide (NO)in the outcome of coxsackievirus B3 (CBV3)-induced acute myocarditis in Se and/or VE-deficient mice as a model of human inflammatory heart muscle disease. Milestone
Substantially Met 2. Clarify the mechanism whereby dietary iron overload in the host leads to severe and unusual genomic mutations in coxsackievirus B3 inoculated into mice deficient in VE. Milestone Not Met Progress slowed by resource limitation (human,fiscal,equipment, etc. 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? Milestone 1 (FY-06)- Several general laboratory procedures including NO assay, glutathione peroxidase (GPX) analysis, and Western blot technique for detection of expressed proteins in tissues of treated animals has been established. Initial experiments will establish resistance of several genetically defined mouse strains to CBV3 infection and NO production will be completed to select an appropriate model system for additional analysis; preparatory procedures for analysis of tissue samples from dietary treated mice will be
analyzed by surface enhanced laser desorption/ionization time-of-flight (SELDI-TOF) to establish patterns of protein expression with and without dietary changes. Milestone 2 (FY-06)- Develop real-time Polymerase Chain Reaction (PCR) procedures; use of the high performance liquid chromatography (HPLC) apparatus; work up the HPLC procedures for peroxynitrite (nitro-tyrosine) and hydoxyl radicals (2,3-dihydroxybenzoic acid) in liver and heart. (FY- 07) Implement VE and liver mineral analysis; analyze samples from pilot study; sequence CVB3 genome from pilot study. Carry out selenosis study; save, process, and start running samples for SELDI-TOF analysis. (FY-08) Conduct any needed replicate studies and prepare data for publication. Milestone 3 (FY-07)-Conduct experiment to determine interactive effects of CVB3 infection and Se deficiency on NO production in vivo; work up analysis for glutathione/oxidized glutathione (GSH/GSSG) ratio in liver and heart. (FY-08) Conduct full experiment on
effect of -VE high Fe diet on CVB3 genome. Analyze data from SELDI-TOF proteomics experiment, and prepare manuscript. Milestone 4 (FY-08)-Analyze data from CVB3 infection and Se deficiency experiment; conduct marginal mono-factorial Se deficiency study. Sequence virus samples from VE high experiment. Verify SELDI-TOF data using 2D acrylamide gel electrophoresis technique. 4a What was the single most significant accomplishment this past year? Dietary restriction increases the pathogenesis of CBV3 infections. Restriction in food intake is gaining greater acceptability by the general public, but the long-term consequences of such a dietary anti- aging strategy is unknown. Our studies with mice show that even a mild- moderate food withdrawal results in a highly undesirable side effect of viral infection (more viral pathogenesis). Thus, the use of food restriction as an approach to a prolonged lifespan appears too dangerous for routine use. 4b List other significant accomplishments, if
any. Several means to increase oxidative stress in vivo also lead to increased virulence of viral infection. We observed that copper deficiency exacerbates infection with the CBV3, presumably by increasing oxidative stress by decreasing the activity of superoxide dismutase. Thus, this is yet another example of how nutritional status can have a profound influence on the ability of a host to resist infection. 4d Progress report. The project is in its second year with progress made in the development of assays to measure selenium-dependent glutathione peroxidase enzyme activity and protein levels by Western blot technology. Real-time PCR to evaluate cell signaling pathways and HPLC methods for detection of vitamin E have been applied to the analysis. Two experiments that related impaired lipid peroxidation induced by copper-deficient changes in oxidative stress (decreased superoxide ismutase) and protein malnutrition (decreased cellular GSH content) were completed and data are under
analysis. Both nutritional manipulations facilitated an increase in CBV3 viral replication showing the importance of redox status in determining the virulence of a viral infection. Detection of a proteomic-fingerprint change in plasma as a result of selenium overexposure suggests that the SELDI-TOF can be applied to the characterization of changes in selenium metabolic patterns that differ during disease or altered nutritional status. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. The work is based on earlier studies by Chinese scientists who had shown that Se supplementation prevented Keshan disease, a cardiomyopathy that afflicts infants or young children who reside in areas with low Se. Collaboration with Professor Ricardo Gomez of the University of Buenos Aires demonstrated that atrial tissue from mice fed a Se-deficient diet for 4 weeks post weaning had a diminished beta-adrenoceptor-inotropic cardiac response to
isoproterenol or norepinephrine compared with atrial tissue from mice fed the same diet supplemented with 0.2 ppm Se. The dose response curves of isoproterenol and norepinephrine were shifted to the right in Se-deficient mice and the potency of the beta-adrenoceptor agonist was diminished. This diminished response could be reversed by pretreating mice with NOS inhibitors such as N-monomethyl-L-arginine or aminopyridine. Elevated serum concentrations of nitrite/nitrate as well as a 3-fold increase in the heart muscle NOS activity was seen in the Se- versus the Se+ mice. Western blotting as well as indirect immunofluorescence indicated an enhanced expression of iNOS in hearts of Se- mice. Increased expression and activity of NOS, and increased nitrite/nitrate levels in Se- mice correlated with an impaired response to beta-adrenergic-isotropic cardiac stimulation. Thus, elevated NO levels due to Se deficiency may account for some of the cardiopathophysiology observed in these
nutritionally-deprived animals. Others have noted the multiple physiological and pathophysiological effects of NO in cardiac function under conditions of nutritional adequacy. In summary, the following experimental observations have been made in the mouse model: 1) Se deficiency leads to increased cardiac production of NO; 2) CVB3 infection leads to increased cardiac production of NO; 3) Se deficiency leads to increased cardiac damage due to CVB3 infection. Therefore, the major goal of the experiments outlined in the project statement is to test the hypothesis that localized increased NO production in the heart in response to infection with CVB3 induces heart muscle damage seen in Se-deficient mice. These studies will have impact on clinical observations of cardiopathology where elements of diet are a factor to be considered. 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? Development of a SELDI-TOF mass spectrometry (MS) analysis of serum and tissues from animals with various levels of Se status will provide a proteomic evaluation that could be used to screen for dietary changes in Se that would be indicative of the appropriateness of Se status for the control of viral infection. These markers could be of additional benefit to other diseases in which Se functions to improve health status such as has been suggested for cancer. This technology, when tested in humans, would be of benefit as a useable marker of Se status. 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). Research cited in article "Getting Serious about Selenium" by Dale Kiefer in December, 2004 issue of Life Extension
magazine. Research highlighted as an example of ARS creativity in an on-line issue of TIME Magazine Special Report: A Half-Century of Invention (October 3, 2004).
Impacts
(N/A)
Publications
- Burk, R.F., Levander, O.A. 2005. Selenium. In: Shils, M.E., Shike, M., Ross, A.C., Caballero, B., Cousins, R.J., editors. Modern Nutrition in Health and Disease. 10th edition. Baltimore, MD: Lippincott Williams & Wilkins. p. 312-325.
- Beck, M.A., Shi, Q., Morris, V.C., Levander, O.A. 2005. Benign coxsackie virus damages heart muscle in iron-loaded vitamin E-deficient mice. Journal of Free Radical Biology and Medicine. 38:(1)112-116.
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