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 Vitamin Metabolism Laboratory investigates the role of folic acid, and vitamins B12 and B6 in health and disease, with a particular focus on the chronic, degenerative diseases that are associated with aging. These vitamins serve as coenzymes in one-carbon metabolism, a network of biochemical reactions essential for normal function. In one-carbon metabolism, a carbon unit, usually from serine, either methylates homocysteine to methionine or is used for purine and thymidylate synthesis. Purines and thymidylate are building blocks for nucleic acids. Methionine is a precursor of S-adenosylmethionine (SAM), the universal methyl donor for many biological compounds, including proteins, lipids, and DNA. Hyperhomocysteinemia, a condition of elevated blood levels of the amino acid homocysteine,
arises from disrupted one-carbon metabolism. Hyperhomocysteinemia has been recently shown to be associated with increased risk of vascular disease, stroke and thrombosis and perhaps dementia and Alzheimer's disease. A major task of this laboratory is to determine the risks and benefits of food folate fortification to the United States (US) population. Folic acid supplementation of flour and grain products was implemented to improve folate status in women of childbearing age and to reduce the incidence of folate-deficiency induced neural tube birth defects. The benefits of this policy for reducing neural tube birth defects are clear; however, the impact on other diseases such as cancer and heart disease, and in other populations, such as children and elders is uncertain. Monitoring of potential health risks in specific vulnerable populations such as older adults or vegans in whom vitamin B12 deficiency is prevalent, and in the general population has been lacking. High folic acid intake
in the presence of B12 deficiency may have harmful effects on the blood and nervous system. Data from our studies will provide the necessary basis for evaluating the impact of food folate fortification policy, and for adjusting folate fortification levels and formulation (i. e., considering the addition of vitamin B12) to optimize health benefits and mitigate potential risks across the population. This research is related to National Program 107 - Human Nutrition, program components: 1. Composition of Foods; 2. Bioavailability of Nutrients and Food Components; 3. Nutrition Monitoring; 4. Nutrient Requirements; 5. Health Promoting Properties of Plant and Animal Foods; and 7. Health Promoting Intervention Strategies for Targeted Populations. 2. List by year the currently approved milestones (indicators of research progress) he Vitamin Metabolism Laboratory investigates the role of folic acid, and vitamins B12 and B6 in health and disease, with a particular focus on the chronic,
degenerative diseases that are associated with aging. These vitamins serve as coenzymes in one-carbon metabolism, a network of biochemical reactions essential for normal function. In one-carbon metabolism, a carbon unit, usually from serine, either methylates homocysteine to methionine or is used for purine and thymidylate synthesis. Purines and thymidylate are building blocks for nucleic acids. Methionine is a precursor of S-adenosylmethionine (SAM), the universal methyl donor for many biological compounds, including proteins, lipids, and DNA. Hyperhomocysteinemia, a condition of elevated blood levels of the amino acid homocysteine, arises from disrupted one-carbon metabolism. Hyperhomocysteinemia has been recently shown to be associated with increased risk of vascular disease, stroke and thrombosis and perhaps dementia and Alzheimer's disease. Milestones: 2005 1. Determine food folic acid content of over 300 food products using the affinity/high performance liquid chromatography
(HPLC) method to assess the amount of folic acid that has been added as a fortificant. Objective 1 2. Determine the relationship between B vitamins, tHcy & methionine and intima-media thickness (IMT), cognitive function and brain atrophy using data collected from the Framingham studies. Objective 2 3. Determine the effect of aging on folate deficiency by using the affinity/HPLC method with electrochemical detection to analyze different tissues from folate deficient and folate-sufficient young and older rats. This information will be used to determine if the combination of aging and restriction of folate intake will affect the types of folate in tissues and whether various tissues are affected differently. Objective 4 4. Distinguish between the possible direct effects of methionine and high homocysteine by using folate-deficient ApoE mice to illicit elevated plasma homocysteine levels, while adding excess vitamins to mice receiving a high methionine diet to suppress the formation of
elevated homocysteine in blood. Objective 5 2006 1. Determine food folic acid content of over 300 food products using the affinity/high performance liquid chromatography (HLPC) method to assess the amount of folic acid that has been added as a fortificant. Objective 1 2. Determine the relationship between B vitamins, tHcy & methionine and intima-media thickness (IMT), cognitive function and brain atrophy using data collected from the Framingham studies. Objective 2 3. Use data collected from the Framingham studies on B vitamin and tHcy content in blood and total methionine intake to determine if incidence of chronic heart disease is related to tHcy, or/and high methionine intake and if so, which of the vitamins measured can be attributed to the increase in tHcy levels and the relationship to the disease. Objective 2 4. Screen 600 men from Milano, Italy and select 20 subjects who are homozygotes for the C677T genotype (TT) and 20 with the wild type (CC) genotype for a study to
determine effect of folate supplementation and the C677T mutation on gene methylation and gene expression. Objective 2 5. Compare data on plasma tHcy and vitamin B12 levels collected from the original Framingham Study Cohort and Offspring Cohort to determine if there are heritable trends in the levels of tHcy and vitamin B12. Objective 3 6. Determine the effect of aging on folate deficiency by using the affinity/HPLC method with electrochemical detection to analyze different tissues from folate deficient and folate-sufficient young and older rats. This information will be used to determine if the combination of aging and restriction of folate intake will affect the types of folate in tissues and whether various tissues are affected differently. Objective 4 7. Distinguish between the possible direct effects of methionine and high homocysteine by using folate-deficient ApoE mice to illicit elevated plasma homocysteine levels, while adding excess vitamins to mice receiving a high
methionine diet to suppress the formation of elevated homocysteine in blood. Objective 5 8. Determine the relationship between B-vitamins vs. homocysteine and behavior in rats made hyperhomocysteinemic by folate deficiency, vitamin B12 deficiency, or a high methionine diet to determine which of these conditions will result in impaired behavior as determined by the Morris Water Maze. Objective 5 2007 1. Use data collected from the analysis of over 300 food products to modify food tables in order to obtain estimates on intake to assess dietary folate equivalents and to determine actual intake of folic acid from supplements and food folate fortification. Objective 1 2. Use a newly devised affinity/HPLC method with electrochemical detection to determine the extent to which higher intake of folic acid is responsible for the presence of unmetabolized folic acid in plasma from Framingham Study subjects and the National Health and Nutrition Examination Survey (NHANES) 1999-2000 population.
Objective 1 3. Use data on B vitamin contents from the original Framingham Study and the NHANES 1999-2000 population to determine if the relationships seen between elevated tHcy and chronic heart failure and incidence of dementia can be attributable to low B vitamin status. Objective 2 4. Determine the effects of placebo versus folate supplementation (400ug/d) on gene expression in 20 subjects who are homozygotes for the C677T genotype (TT) and 20 with the wild type (CC) genotype. Objective 2 5. Compare data collected on tHcy in both the original Framingham Study cohort and offspring cohort to determine the interaction between tHcy and genetic background on CVD. Objective 3 6. Determine the effect of B-vitamins and homocysteine on behavior of rats made hyperhomocysteinemic by folate deficiency, vitamin B12 deficiency or high methionine diet. Objective 5 7. Determine the effect of excess folate in rats maintained on a B12- deficient diet for an extended time (more than 6 months) and
fed an excess (8-16-fold the RDA) of folic acid. Assess whether administration of high levels of folic acid exacerbates the signs of B12 deficiency (anemia and neurological insufficiency). Objective 5. 2008 1. Use a newly devised affinity/HPLC method with electrochemical detection to determine the extent to which a higher intake of folic acid is responsible for the presence of unmetabolized folic acid in plasma from Framingham Study subjects and the NHANES 1999-2000 population. Objective 1 2. Use the data on unmetabolized folic acid collected in the Framingham Study and the NHANES 1999-2000 population to determine if highunmetabolized folic acid level is associated with a lower cognitive function in the elderly. Objective 1 3. Determine the effect of folate supplementation (400 mcg/day) on gene methylation and gene expression among 20 subjects who are homozygotes for the C677T genotype (TT) and 20 with the wild type (CC) genotype using microarray and other molecular biology methods.
Objective 2 4. Compare data collected on tHcy in both the original Framingham Study cohort and offspring cohort to determine the interaction between tHcy and genetic background on CVD. Objective 3 5. Compare data collected on tHcy in both the Framingham Study original cohort and offspring cohort to determine the extent of heritability of the relationship between tHcy and cognitive function, using family linkages. Objective 3 6. Use rats made hyperhomocysteinemic by folate deficiency, vitamin B12 deficiency, or a high methionine diet to determine which of these conditions results in impaired behavior as determined by the Morris Water Maze. Objective 5 7. Cross the ApoE-null mouse and the CBS-deficient mouse to obtain a model that is susceptible to neurological degeneration and elevated plasma tHcy levels to determine if high homocysteine per se produces neurological decline. Objective 5 8. Observe rats maintained on a B12-deficient diet for an extended time (more than 6 months) and fed
with excess (8-16- fold the RDA) folic acid to determine if the administration of high levels of folic acid exacerbates the signs of B12 deficiency (anemia and neurological insufficiency). Objective 5 2009 1. Use the data on unmetabolized folic acid collected in the Framingham Study to determine if high unmetabolized folic acid level is associated with a lower cognitive function in the elderly. Objective 1 2. Determine the effect of folate supplementation (400ug/d) on gene methylation and gene expression among 20 subjects who are homozygotes for the C677T genotype (TT) and 20 with the wild type (CC) genotype using microarray and other molecular biology methods. Objective 2 3. Compare data collected on tHcy in both the original Framingham Study cohort and offspring cohort to determine the interaction between tHcy and genetic background on CVD. Objective 3. 4. Cross the ApoE-null mouse and the CBS deficient mouse to obtain a model that is susceptible to neurological degeneration and
elevated plasma tHcy levels to determine if high homocysteine per se produces neurological decline. Objective 5. 5. Observe rats maintained on a B12-deficient diet for an extended time (more than 6 months) and fed with excess (8-16- fold the RDA) folic acid to determine if the administration of high levels of folic acid exacerbates the signs of B12 deficiency (anemia and neurological insufficiency). Objective 5. This research is related to National Program 107 Human Nutrition, program components: 4. Nutrient Requirements; 7. Health Promoting Intervention Strategies for Targeted Populations. 4a List the single most significant research accomplishment during FY 2006. Folate Intake and Natural Killer (NK) Cell Activity In collaboration with scientists from the Huntington Cancer Center in Seattle, Washington, we determined whether a higher intake of folate by postmenopausal women compromises natural killer (NK) cell activity. We used our recently developed affinity/HPLC method with
electrochemical detection to show that: 1) unmetabolized folic acid is present in the plasma of almost 80% of these women. 2) NK activity in women over the age of 60 years exhibited an inverse correlation with concentration of unmetabolized folic acid. 3) There was no correlation between NK activity and total folate, total 5-methyltetrahydrofolate (which comprised the predominant form of plasma folate), in women below the age of 60. This is the first report to focus on the need to study the potential benefits and risks of excessive folic acid intake. A major task of this laboratory s to determine the risks and benefits of food folate fortification to the United States (US) population. Folic acid supplementation of flour and grain products was implemented to improve folate status in women of childbearing age and to reduce the incidence of folate-deficiency induced neural tube birth defects. The benefits of this policy for reducing neural tube birth defects are clear; however, the
impact on other diseases such as cancer and heart disease, and in other populations, such as children and elders is uncertain. Monitoring of potential health risks in specific vulnerable populations such as older adults or vegans in whom vitamin B12 deficiency is prevalent, and in the general population has been lacking. High folic acid intake in the presence of B12 deficiency may have harmful effects on the blood and nervous system. Data from our studies will provide the necessary basis for evaluating the impact of food folate fortification policy, and for adjusting folate fortification levels and formulation (i. e., considering the addition of vitamin B12) to optimize health benefits and mitigate potential risks across the population. This research is related to National Program 107-Human Nutrition, program components: 4. Nutrient Requirements; and 7. Health Promoting Intervention Strategies for Targeted Populations. 5. Describe the major accomplishments to date and their predicted
or actual impact. The relationship between B-vitamin metabolism and impairments that afflict the elderly is an expanding research area. Many of these relationships have been identified through epidemiological studies. The Vitamin Metabolism Laboratory is working to identify new relationships and determine the mechanisms that underlie these relationships. In demonstrating and elucidating the relation of B- vitamin and homocysteine metabolism to the aging process, in both animal models and in epidemiological studies, our work is instrumental for improving nutrition and implementing nutritional and other interventions aimed at reducing the risk and burden of age-related diseases such as cancer, heart disease and dementia. In determining food folic acid content, our studies will provide the necessary basis for evaluating the impact of food folate fortification policy, and for adjusting folate fortification levels and formulation (i.e., considering the addition of vitamin B12) to optimize
health benefits and mitigate potential risks across the population. We used the affinity/HPLC method developed in our laboratory to conduct a survey of 370 food products that were supplied to us by the USDA Human Nutrition Research Center in Beltsville, MD, to determine amount of folic acid that has been added as a fortificant. We have begun to incorporate these data into the database of food intake for the Framingham Study and are in the process of sending these data to the USDA to revise the food folate tables. (Objective 1, Milestone 1) We made progress in determining the relationship between B vitamins, total homocysteine (tHcy) and methionine and intima-media thickness (IMT), cognitive function and brain atrophy using data collected from the Framingham studies. (Objective 2, Milestone 2) We used data collected from the Framingham studies on B vitamin and tHcy content in blood and total methionine intake to determine if incidence of chronic heart disease is related to tHcy,
or/and high methionine intake and if so, which of the vitamins measured can be attributed to the increase in tHcy levels and the relationship to the disease. (Objective 2, Milestone 3) We began the screening of 600 men from Milano, Italy and will select 20 subjects who are homozygotes for the C677T genotype (TT) and 20 with the wild type (CC) genotype for a study to determine the effect of folate supplementation and the C677T mutation on gene methylation and gene expression. (Objective 2, Milestone 4) We have determined the effect of aging on folate deficiency by using the affinity/HPLC method with electrochemical detection to analyze livers from folate deficient and folate-sufficient young and older rats. This information will be used to determine if the combination of aging and restriction of folate intake will affect the types of folate in tissues and whether various tissues are affected differently. (Objective 4, Milestone 6) We studied the possible direct effects of methionine
and highhomocysteine by using folate-deficient ApoE mice to illicit elevated plasma homocysteine levels, while adding excess vitamins to mice receiving a high methionine diet to suppress the formation of elevated homocysteine in blood. (Objective 5 , Milestone 7) We determined the relationship between B-vitamins vs. homocysteine and behavior in rats made hyperhomocysteinemic by folate deficiency, vitamin B12 deficiency, or a high methionine diet to determine which of these conditions will result in impaired behavior as determined by the Morris Water Maze. (Objective 5 , Milestone 8) We have completed the first series of analyses using livers from young and older rats that were supplemented with a folate-deficient and folate-sufficient diet. (Objective 4, Milestone 3) These accomplishments are related to National Program 107 Human Nutrition program components: 1. Composition of Foods; 2. Bioavailability of Nutrients and Food Components; 3. Nutrition Monitoring; 4. Nutrient
Requirements; 5. Health Promoting Properties of Plant and Animal Foods; and 7. Health Promoting Intervention Strategies for Targeted Populations. 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? Our research on B vitamin nutrition and cardiovascular and other age- related diseases has been reported in peer-reviewed journals and has been presented to industry groups and scientists at professional meetings. The leader of this CRIS project was invited to present three papers that discuss methods of estimating folate and B12 requirements at the World Health Organizations Technical Consultation on Folate and Vitamin B-12 Deficiencies in Geneva, Switzerland, October 17-21, 2005His recommendations will be instrumental in setting up a micronutrient
fortification programs worldwide 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). World Health Organizations Technical Consultation on Folate and Vitamin B-12 Deficiencies, Geneva, Switzerland - October 17-21, 2005. Jacob Selhub presented three lectures: 1. Public health significance of fortification of grain products with folic acid 2. Public health significance of elevated homocysteine 3. Indicators of folate and vitamin B12 status. Jacob Selhub, Renal folate absorption and the kidney binding protein, presented at the 1st International Workshop on Folate Receptors. June 11- 15, 2006
Impacts
(N/A)
Publications
- Lim, U., Peng, K., Shane, B., Stover, P.J., Litonjua, A.A., Weiss, S.T., Gaziano, M.J., Strawderman, R.L., Raiszadeh, F., Selhub, J., Tucker, K.L., Cassano, P.A. 2005. Polymorphisms in cytoplasmic serine hydroxymethyltransferase and methylenetetrahydrofolate reductase affect the risk of cardiovascular disease in men. Journal of Nutrition. 135(8) :1989-94.
- Selhub, J. 2006. The many facets of hyperhomocysteinemia: studies from the framingham cohorts. Journal of Nutrition. 136(6 Suppl):1726S-1730S.
- Anguera, M.C., Ghandour, H., Chiang, E., Selhub, J., Shane, B., Stover, P. J. 2006. Regulation of folate-mediated one-carbon metabolism by 10- formyltetrahydrofolate dehydrogenase. Journal of Biological Chemistry. 281(27):18335-42.
- Troen, A.M., Mitchell, B., Sorensen, B., Wener, M.H., Johnston, A., Wood, B., Selhub, J., Mctiernan, A., Yasui, Y., Oral, E., Potter, J.D., Ulrich, C.M. 2006. Unmetabolized folic acid in plasma is associated with reduced natural killer cell cytotoxicity among postmenopausal women. Journal of Nutrition. 136(1):189-94.
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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? The Vitamin Metabolism Laboratory investigates the role of folic acid, and vitamins B12 and B6 in health and disease, with a particular focus on the chronic, degenerative diseases that are associated with aging. These vitamins serve as coenzymes in one-carbon metabolism, a network of biochemical reactions essential for normal function. In one-carbon metabolism, a carbon unit, usually from serine, either methylates homocysteine to methionine or is used for purine and thymidylate synthesis. Purines and thymidylate are building blocks for nucleic acids. Methionine is a precursor of S-adenosylmethionine (SAM), the universal methyl donor for many biological compounds, including proteins, lipids, and DNA. Hyperhomocysteinemia, a condition of elevated blood levels of the amino acid homocysteine, arises from
disrupted one carbon metabolism. Hyperhomocysteinemia has been recently shown to be associated with increased risk of vascular disease, stroke and thrombosis and perhaps dementia and Alzheimer's disease. A major task over the next 4 years is to determine the risks and benefits of food folate fortification to the US population. Folic acid supplementation of flour and grain products was implemented to improve folate status in women of childbearing age and to reduce the incidence of folate-deficiency induced neural tube birth defects. The benefits of this policy for reducing neural tube birth defects are clear; however, the impact on other diseases such as cancer and heart disease, and in other populations, such as children and elders in uncertain. Monitoring of potential health risks in specific vulnerable populations such as older adults or vegans in whom vitamin B12 deficiency is prevalent, and in the general population has been lacking. High folic acid intake in the presence of B12
deficiency may have harmful effects on the blood and nervous system. Data from our studies will provide the necessary basis for evaluating the impact of food folate fortification policy, and for adjusting folate fortification levels and formulation (i.e., considering the addition of vitamin B12) to optimize health benefits and mitigate potential risks across the population. This research is related to National Program 107 - Human Nutrition, program components: 1.Composition of Foods; 2. Bioavailability of Nutrients and Food Components; 3. Nutrition Monitoring; 4. Nutrient Requirements; 5. Health Promoting Properties of Plant and Animal Foods; and 7.Health Promoting Intervention Strategies for Targeted Populations 2. List the milestones (indicators of progress) from your Project Plan. Objective 1: To determine the impact of mandatory food folic acid fortification in the United States (US). Objective 2: Determine the interrelationships between B vitamin status, methionine intake,
genetic polymorphism and plasma homocysteine (tHcy). Objective 3: Determine the hereditary association of plasma homocysteine and vitamin status. Objective 4: Determine the impact of aging on one-carbon metabolism in rats by measuring folate form distribution. Objective 5: Determine the biochemical, pathological and functional impact of nutritional and genetic disruptions of one-carbon metabolism, in animal models of age-related vascular and neurological dysfunction, with emphasis on the roles of B-vitamins, homocysteine and methionine in tissue-specific susceptibility to disease. Milestones: 2005 1. Determine food folic acid content of over 300 food products using the affinity/high performance liquid chromatography (HPLC) method to assess the amount of folic acid that has been added as a fortificant. Objective 1 2. Determine the relationship between B vitamins, tHcy & methionine and intima-media thickness (IMT), cognitive function and brain atrophy using data collected from the
Framingham studies. Objective 2 3. Determine the effect of aging on folate deficiency by using the affinity/HPLC method with electrochemical detection toanalyze different tissues from folate-deficient and folate-sufficient young and older rats. This information will be used to determine if the combination of aging and restriction of folate intake will affect the types of folate in tissues and whether various tissues are affected differently. Objective 4 4. Distinguish between the possible direct effects of methionine and high homocysteine by using folate-deficient ApoE mice to illicit elevated plasma homocysteine levels, while adding excess vitamins to mice receiving a high methionine diet to suppress the formation of elevated homocysteine in blood. Objective 5 2006 1. Determine food folic acid content of over 300 food products using the affinity/high performance liquid chromatography (HLPC) method to assess the amount of folic acid that has been added as a fortificant. Objective 1 2.
Determine the relationship between B vitamins, tHcy & methionine and intima-media thickness (IMT), cognitive function and brain atrophy using data collected from the Framingham studies. Objective 2 3. Use data collected from the Framingham studies on B vitamin and tHcy content in blood and total methionine intake to determine if incidence of chronic heart disease is related to tHcy, or/and high methionine intake and if so, which of the vitamins measured can be attributed to the increase in tHcy levels and the relationship to the disease. Objective 2 4. Screen 600 men from Milano, Italy and select 20 subjects who are homozygotes for the C677T genotype (TT) and 20 with the wild type (CC) genotype for a study to determine effect of folate supplementation and the C677T mutation on gene methylation and gene expression. Objective 2 5. Compare data on plasma tHcy and vitamin B12 levels collected from the original Framingham Study Cohort and Offspring Cohort to determine if there are
heritable trends in the levels of tHcy and vitamin B12. Objective 3 6. Determine the effect of aging on folate deficiency by using the affinity/HPLC method with electrochemical detection to analyze different tissues from folate-deficient and folate-sufficient young and older rats. This information will be used to determine if the combination of aging and restriction of folate intake will affect the types of folate in tissues and whether various tissues are affected differently. Objective 4 7. Distinguish between the possible direct effects of methionine and high homocysteine by using folate-deficient ApoE mice to illicit elevated plasma homocysteine levels, while adding excess vitamins to mice receiving a high methionine diet to suppress the formation of elevated homocysteine in blood. Objective 5 8. Determine the relationship between B-vitamins vs. homocysteine and behavior in rats made hyperhomocysteinemic by folate deficiency, vitamin B12 deficiency, or a high methionine diet to
determine which of these conditions will result in impaired behavior as determined by the Morris Water Maze. Objective 5 2007 1. Use data collected from the analysis of over 300 food products to modify food tables in order to obtain estimates on intake to assess dietary folate equivalents and to determine actual intake of folic acid from supplements and food folate fortification. Objective 1 2. Use a newly devised affinity/HPLC method with electrochemical detection to determine the extent to which higher intake of folic acid is responsible for the presence of unmetabolized folic acid in plasma from Framingham Study subjects. Objective 1 3. Use data on B vitamin contents from the original Framingham Study to determine if the relationships seen between elevated tHcy and chronic heart failure and incidence of dementia can be attributable to low B vitamin status. Objective 2 4. Determine the effects of placebo versus folate supplementation (400ug/d) on gene expression in 20 subjects who
are homozygotes for the C677T genotype (TT) and 20 with the wild type (CC) genotype. Objective 2 5. Compare data collected on tHcy in both the original Framingham Study cohort and offspring cohort to determine the interaction between tHcy and genetic background on CVD. Objective 3 6. Determine the effect of B-vitamins and homocysteine on behavior of rats made hyperhomocysteinemic by folate deficiency, vitamin B12 deficiency or high methionine diet. Objective 5 7. Determine the effect of excess folate in rats maintained on a B12- deficient diet for an extended time (more than 6 months) and fed an excess (8-16-fold the RDA) of folic acid. Assess whether administration of high levels of folic acid exacerbates the signs of B12 deficiency (anemia and neurological insufficiency). Objective 5. 2008 1. Use a newly devised affinity/HPLC method with electrochemical detection to determine the extent to which a higher intake of folic acid is responsible for the presence of unmetabolized folic
acid in plasma from Framingham Study subjects. Objective 1 2. Use the data on unmetabolized folic acid collected in the Framingham Study to determine if high unmetabolized folic acid level is associated with a lower cognitive function in the elderly. Objective 1 3. Determine the effect of folate supplementation (400ug/d) on gene methylation and gene expression among 20 subjects who are homozygotes for the C677T genotype (TT) and 20 with the wild type (CC) genotype using microarray and other molecular biology methods. Objective 2 4. Compare data collected on tHcy in both the original Framingham Study cohort and offspring cohort to determine the interaction between tHcy and genetic background on CVD. Objective 3 5. Compare data collected on tHcy in both the Framingham Study original cohort and offspring cohort to determine the extent of heritability of the relationship between tHcy and cognitive function, using family linkages. Objective 3 6. Use rats made hyperhomocysteinemic by
folate deficiency, vitamin B12 deficiency, or a high methionine diet to determine which of these conditions results in impaired behavior as determined by the Morris Water Maze. Objective 5 7. Cross the ApoE-null mouse and the CBS-deficient mouse to obtain a model that is susceptible to neurological degeneration and elevated plasma tHcy levels to determine if high homocysteine per se produces neurological decline. Objective 5 8. Observe rats maintained on a B12-deficient diet for an extended time (more than 6 months) and fed with excess (8-16- fold the RDA) folic acid to determine if the administration of high levels of folic acid exacerbates the signs of B12 deficiency (anemia and neurological insufficiency). Objective 5 2009 1. Use the data on unmetabolized folic acid collected in the Framingham Study to determine if high unmetabolized folic acid level is associated with a lower cognitive function in the elderly. Objective 1 2. Determine the effect of folate supplementation (400ug/d)
on gene methylation and gene expression among 20 subjects who are homozygotes for the C677T genotype (TT) and 20 with the wild type (CC) genotype using microarray and other molecular biology methods. Objective 2 3. Compare data collected on tHcy in both the original Framingham Study cohort and offspring cohort to determine the interaction between tHcy and genetic background on CVD. Objective 3. 4. Cross the ApoE-null mouse and the CBS deficient mouse to obtain a model that is susceptible to neurological degeneration and elevated plasma tHcy levels to determine if high homocysteine per se produces neurological decline. Objective 5. 5. Observe rats maintained on a B12-deficient diet for an extended time (more than 6 months) and fed with excess (8-16- fold the RDA) folic acid to determine if the administration of high levels of folic acid exacerbates the signs of B12 deficiency (anemia and neurological insufficiency). Objective 5. 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. Vitamin Metabolism Laboratory Milestone # 1 Determine food folic acid content of over 300 food products using the affinity/high performance liquid chromatography (HPLC) method to assess the amount of folic acid that has been added as a fortificant. Objective 1. Milestone Substantially Met 2. Vitamin Metabolism Laboratory Milestone # 2 Determine the relationship between B vitamins, tHcy & methionine and intima-media thickness (IMT), cognitive function and brain atrophy using data collected from the Framingham studies. Objective 2. Milestone Substantially Met 3. Vitamin Metabolism Laboratory Milestone # 3 Determine the effect of aging on folate deficiency by using the affinity/HPLC method with electrochemical detection to analyze different tissues from folate-deficient and folate-sufficient young and older rats. This information will be used to determine if the
combination of aging and restriction of folate intake will affect the types of folate in tissues and whether various different tissues are affected differently. Objective 4. Milestone Substantially Met 4. Vitamin Metabolism Laboratory Milestone # 4 Distinguish between the possible direct effects of methionine and high homocysteine by using folate-deficient ApoE mice to illicit elevated plasma homocysteine levels, while adding excess vitamins to mice receiving a high methionine diet to suppress the formation of elevated homocysteine in blood. Objective 5. 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? 2006 1. Determine food folic acid content of over 300 food products using the affinity/high performance liquid chromatography (HLPC) method to assess the amount of folic acid that has been added as a fortificant.
Objective 1 Expected results: The dietary folate equivalent (DFE) has been instilled to reflect the higher bioavailability of folic acid than naturally occurring food folates. Measuring folic acid in foods will allow the modification of food folate tables for estimation of DFE intake. 2. Determine the relationship between B vitamins, tHcy & methionine and intima-media thickness (IMT), cognitive function and brain atrophy using data collected from the Framingham studies. Objective 2 Expected results: In the past, research has been directed toward determining relationship of IMT, cognitive function and brain atrophy with homocysteine. Our expectation is that some of these diseases will also be related to determinants of plasma homocysteine, i.e., increased methionine intake, low status of folate, B12 or all of the above. 3. Use data collected from the Framingham studies on B vitamin and tHcy content in blood and total methionine intake to determine if incidence of chronic heart disease
is related to tHcy, or/and high methionine intake and if so, which of the vitamins measured can be attributed to the increase in tHcy levels and the relationship to the disease. Objective 2 Expected results: We have shown previously that chronic heart failure is associated with elevated plasma homocysteine levels. We expect that this relationship is at least partially attributed to low status of folate and/or vitamin B12. 4. Screen 600 men from Milano, Italy and select 20 subjects who are homozygotes for the C677T genotype (TT) and 20 with the wild type (CC) genotype for a study to determine effect of folate supplementation and the C677T mutation on gene methylation and gene expression. Objective 2. Expected results: Complete screening and enrollment of the subjects for this study. 5. Compare data on plasma tHcy and vitamin B12 levels collected from the original Framingham Study Cohort and Offspring Cohort to determine if there are heritable trends in the levels of tHcy and vitamin
B12. Objective 3. Expected Results: The relationship of homocysteine and vitamin B12 concentrations in plasma will be similar in the offspring and parent groups. This similarity is the reflection of the concentration of both analytes in plasma resulting from both environmental and genetic factors. 6. Determine the effect of aging on folate deficiency by using the affinity/HPLC method with electrochemical detection to analyze different tissues from folate-deficient and folate-sufficient young and older rats. This information will be used to determine if the combination of aging and restriction of folate intake will affect the types of folate in tissues and whether various tissues are affected differently. Objective 4. Expected Results: Folate forms distribution will be altered in the aged folate-deficient rat compared to the folate-deficient younger rat. This change will reflect a lower capacity of the older rat to accomplish methylation reactions. 7. Distinguish between the possible
direct effects of methionine and high homocysteine by using folate-deficient ApoE null mice to illicit elevated plasma homocysteine levels, while adding excess vitamins to mice receiving the high methionine diet to suppress the formation of elevated homocysteine in blood. Objective 5 Expected Results: Intake of excess methionine will result in increased CVD in the ApoE null mice. This will be accompanied by an increase in inflammation markers in the aorta as determined by an increase in messengers for TNF alpha, VCAM, iNOS and the respective proteins. Increased plasma homocysteine induced by vitamin deficiencies will have less significant effects. 8. Determine the relationship between B-vitamins vs. homocysteine and behavior in rats made hyperhomocysteinemic by folate deficiency, vitamin B12 deficiency, or a high methionine diet to determine which of these conditions will result in impaired behavior as determined by the Morris Water Maze. Expected Results: In the ApoE null mice we
found that vitamin-induced hyperhomocysteinemia is associated with diminished learning behavior. These experiments will show that it is the folate deficiency, not the high homocysteine that causes this altered behavior. 2007 1. Use data collected from the analysis of over 300 food products to modify food tables in order to obtain estimates on intake to assess dietary folate equivalents and to determine actual intake of folic acid from supplements and food folate fortification. Objective 1. Expected Results: The use of the modified food folate intake data based on the DFE will provide a better assessment of folate intake, which is necessary to determine impact of folate in health and disease. 2. Use a newly devised affinity/HPLC method with electrochemical detection to determine the extent to which higher intake of folic acid is responsible for the presence of unmetabolized folic acid in plasma from Framingham Study subjects. Objective 1 Expected Results: We expect that a high
proportion of Americans will have unmetabolized folic acid in their blood. The concentration of folic acid in plasma will increase with increased dietary folic acid intake and will also be dependent on other factors that are yet to be determined. 3. Use data on B vitamin contents from the original Framingham Study to determine if the relationships seen between elevated tHcy and chronic heart failure and incidence of dementia can be attributable to low B vitamin status. Objective 2 Expected Results: We have shown previously that chronic heart failure and dementia are associated with elevated plasma homocysteine levels. We expect that these relationships are at least partially due to low status of folate and/or vitamin B12. 4. Determine the effects of placebo versus folate supplementation (400ug/d) on gene expression in 20 subjects who are homozygotes for the C677T genotype (TT) and 20 with the wild type (CC) genotype. Objective 2 Expected Results: The C677T mutation in the MTHFR gene
was shown to be expressed at low folate status. Under these conditions DNA methylation will be greatly reduced. Gene array analysis of cDNA prepared from RNA of these subjects will show that the expression of certain genes will be altered by this mutation and by folate status. 5. Compare data collected on tHcy in both the original Framingham Study cohort and offspring cohort to determine the interaction between tHcy and genetic background on CVD. Objective 3 Expected Results: Elevated plasma homocysteine levels in offspring will be a predictor of coronary vascular disease in parents. This relationship is a reflection of the fact that plasma levels of homocysteine are in part genetically determined. 6. Determine the effect of B-vitamins and Homocysteine on behavior of rats made hyperhomocysteinemic by folate deficiency, vitamin B12 deficiency or high methionine diet. Objective 5 Expected Results: In the ApoE null mice we found that vitamin-induced hyperhomocysteinemia is associated
with diminished learning behavior. These experiments will show that it is folate deficiency, not a high homocysteine level that causes this altered behavior. 7. Determine the effect of excess folate in rats maintained on a B12- deficient diet for an extended time (more than 6 months) and fed an excess (8-16-fold the RDA) of folic acid. Assess whether administration of high levels of folic acid exacerbates the signs of B12 deficiency (anemia and neurological insufficiency). Objective 5 Expected Results: Excess intake of folic acid in B12-deficient rats will be associated with neurological manifestations and impaired brain function. This association would be similar to what has been referred to in man as: the masking of the neurological manifestations of vitamin B12- related pernicious anemia by excess intake of folic acid. 2008 1. Use a newly devised affinity/HPLC method with electrochemical detection to determine the extent to which higher intake of folic acid is responsible for the
presence of unmetabolized folic acid in plasma from Framingham Study subjects. Objective 1 Expected Results: We expect that a high proportion of Americans will have unmetabolized folic acid in their blood. The concentration of folic acid in plasma will increase with increased dietary folic acid intake and will also be dependent on other factors that are yet to be determined. 2. Use the data on unmetabolized folic acid collected in the Framingham Study to determine if high unmetabolized folic acid level is associated with a lower cognitive function in the elderly. Objective 1 Expected Results: Continuous presence of high concentrations of unmetabolized folic acid will be associated with impaired cognitive function in those elderly subjects whose vitamin B12 status is marginal 3. Determine the effect of folate supplementation (400ug/d) on gene methylation and gene expression among 20 subjects who are homozygotes for the C677T genotype (TT) and 20 with the wild type (CC) genotype using
microarray and other molecular biology methods. Objective 2 Expected Results: The C677T mutation in the MTHFR gene has been shown to be expressed at low folate status. Under these conditions DNA methylation is considerably reduced. Gene array analysis of cDNA prepared from RNA of these subjects will show that the expression of certain genes will be altered by this mutation and by the folate status. 4. Compare data collected on tHcy in both the original Framingham Study cohort and offspring cohort to determine the interaction between tHcy and genetic background on CVD. Objective 3 Expected Results: Elevated plasma homocysteine levels in offspring will be a predictor of coronary vascular disease in parents. This relationship is a reflection of the fact that plasma homocysteine levels are in part genetically determined. 5. Compare data collected on tHcy in both the Framingham Study original cohort and offspring cohort to determine the extent of heritability of the relationship between
tHcy and cognitive function, using family linkages. Objective 3 Expected Results: Elevated plasma homocysteine levels in offspring will be a predictor of cognitive function in parents. This relationship is a reflection of the fact that plasma homocysteine levels are in part genetically determined. 6. Use rats made hyperhomocysteinemic by folate deficiency, vitamin B12 deficiency, or a high methionine diet to determine which of these conditions results in impaired behavior as determined by the Morris Water Maze. Objective 5. Expected Results: In the ApoE null mice we found that vitamin-induced hyperhomocysteinemia is associated with diminished learning behavior. These experiments will show that it the folate deficiency, not the high homocysteine that causes this altered behavior. 7. Cross the ApoE-null mouse and the CBS deficient mouse to obtain a model that is susceptible to neurological degeneration and elevated plasma tHcy levels to determine if high homocysteine per se produces
neurological decline. Objective 5. Expected Results: In the ApoE null mice we found that vitamin-induced hyperhomocysteinemia is associated with diminished learning behavior. The cross between ApoE-null and CBS-deficient mouse will be associated with elevated plasma homocysteine levels. This mouse will behave similar to its parents, since impaired behavior is caused by folate deficiency, not by high homocysteine levels. 8. Observe rats maintained on a B12-deficient diet for an extended time (more than 6 months) and fed with excess (8-16- fold the RDA) folic acid to determine if the administration of high levels of folic acid exacerbates the signs of B12 deficiency (anemia and neurological insufficiency). Objective 5. Expected Results: Excess intake of folic acid in B12-deficient rats will be associated with neurological manifestations and impaired brain function. This association would be similar to what has been referred to in man as: the masking of the neurological manifestations of
vitamin B12- related pernicious anemia by excess intake of folic acid. 4a What was the single most significant accomplishment this past year? A collaborative study that was begun during the last CRIS cycle with researchers from University Hospital in Aachen, Germany and SUNY Downstate Medical Center in Brooklyn, NY, has shown that the presence of circulating autoantibodies against the folate receptor is the cause for cerebral folate deficiency in children. A folate receptor in the choroids plexus is responsible for folate transport in the brain; hence the circulating autoantibodies inhibit the binding of folate by the receptors and impair folate transport across the blood brain barrier. We hypothesize that this is not an isolated phenomenon and that circulating autoantibodies are likely to be prevalent in the population and may explain at least in part some of the folate-dependent impairment of brain functions in the elderly, such as decline in cognitive function, and dementia. 5.
Describe the major accomplishments over the life of the project, including their predicted or actual impact. The relationship between B-vitamin metabolism and impairments that afflict the elderly is an expanding research area. Many of these relationships have been identified through epidemiological studies. The Vitamin Metabolism Laboratory is working to identify new relationships and determine the mechanisms that underlie these relationships. In demonstrating and elucidating the relation of B-vitamin and homocysteine metabolism to the aging process, in both animal models and in epidemiological studies, our work is instrumental for improving nutrition amd implementing nutritional and other interventions aimed at reducing the risk and burden of age related diseases such as cancer heart disease and dementia. In determining food folic acid content, our studies will provide the necessary basis for evaluating the impact of food folate fortification policy, and for adjusting folate
fortification levels and formulation (i.e., considering the addition of vitamin B12) to optimize health benefits and mitigate potential risks across the population. Over the life of the project scientists have used the affinity/HPLC method developed in our laboratory to conduct a survey of 370 food products that were supplied to us by the USDA in Beltsville, to determine amount of folic acid which has been added as a fortificant. We have begun to incorporate these data into the database of food intake for the Framingham Study and are in the process of sending these data to the USDA to revise the food folate tables. (Objective 1, Milestone 1) We have begun data analyses of the relationship of tHcy and B vitamins and methionine intake to IMT in the Framingham Study Offspring cohort. (Objective 2, Milestone 2) We have completed the first series of analyses using livers from young and older rats that were supplemented with a folate- deficient and folate- sufficient diet. (Objective 4,
Milestone 3) We have completed the first series studies on effect of high methionine vs. low vitamin diets on vascular disease. (Objective 5, Milestone 4) These accomplishments are related to National Program 107 - Human Nutrition program components: 1. Composition of Foods; 2. Bioavailability of Nutrients and Food Components; 3. Nutrition Monitoring; 4. Nutrient Requirements; 5. Health Promoting Properties of Plant and Animal Foods; and 7. Health Promoting Intervention Strategies for Targeted Populations; and to ARS Performance Measure 4.1.2 Improve Human Health by Better Understanding the Nutrient Requirements of Individuals and the Nutritional Value of Foods. 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? Our research on B vitamin nutrition and
cardiovascular and other age- related diseases has been reported in peer-reviewed journals and has been presented to industry groups and scientists at professional meetings. 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. Selhub. Plasma Homocysteine Levels: Nutritional and Genetic Regulations and Relationship to CNS Disorders and Alzheimer's June 20-24, 2004. XXIVth CINP Congress. Paris, France June 20-24, 2004 Approaches for Intervention and Impacts on Populations in Israel and Palestine. Child Nutrition in Israel and Palestine Jacob Selhub, Beersheva, Israel, February 2005 J. Selhub. Food Fortification with Micronutrients Needs of Food Monitoring. May 23, 2005. Strategies for the prevention of micronutrients deficiencies. Jerusalem, Israel J. Selhub. Folate fortification. Effect on folate status and homocysteine in the Framingham Study.
13th International Symposium on Chemistry and Biology of Pteridines and Folates Egmond aan Zee, The Netherlands, June 20-24, 2005 Selhub, J; the many facets of hyperhomocysteinemnia: Presidential Plenary Lecture: Homocysteine Metabolism - 5th International Conference Milano, Italy, June 26-30, 2005. Troen AM. Homocysteinemia and Cognitive Dysfunction: Insight from Animal Models. Plenary Lecture: Homocysteine Metabolism - 5th International Conference Milano, Italy, June 26-30, 2005. Troen AM, Scott T, D'Anci KE, Jacques PF, Selhub J, Rosenberg IH and the FAVORIT trial consortium. (2005). Cognitive Function and Homocysteine in Renal Transplant Recipients: A FAVORIT ancillary study - Design and Cohort Description. Hematologica Reports 1(3) p.21. Homocysteine Metabolism - 5th International Conference Milano, Italy, June 26-30, 2005. Troen AM, D'Anci KE, Albuquerque BM, Smith DE, Selhub J, Rosenberg IH, Kruger WD. (2005) Behavioral phenotype of adult-onset homocysteinemia in aged mice with
conditional CBS deficiency. Hematologica Reports 1(3) p.28. Homocysteine Metabolism - 5th International Conference Milano, Italy, June 26-30, 2005. Troen AM, D'Anci KE, Albuquerque BM, Smith DE, Rosenberg IH, Selhub J (2005). Folate-deficiency-induced cognitive deficits in rat are ameliorated by methionine and are unrelated to homocysteinemia. Hematologica Reports 1(3) p.51. Homocysteine Metabolism - 5th International Conference Milano, Italy, June 26-30, 2005.
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