Research Project: One Carbon Nutrients and Metabolism

Location: Jean Mayer Human Nutrition Research Center On Aging

2015 Annual Report

Objectives
LAB NAME: Vitamin Metabolism & Aging 1. Develop new and efficient methods for assessing nutritional status in individuals for B vitamins and their functional markers with internal standards obtained from plants grown on D2O or yeast grown on C-13 glucose. 2. Determine the biochemical, pathological and functional impact of nutritional status and genetic variations in B complex vitamin metabolism with special emphasis on the effect of age, in human, animal and cell culture models. Consider the nutritional status of other B vitamins while studying the effect of each B vitamin. 3. Determine the impact of mandatory folic acid fortification of cereals including effects of improved nutritional status as well as excess intake of folic acid on outcomes including disease risk, cognitive function, inflammation and immune response, with emphasis on understanding the mechanism behind the side-effects.

Approach
LAB NAME: Vitamin Metabolism & Aging We will use biochemical, molecular biological and epidemiological approaches to study the role of B vitamins and the genes involved in their metabolism, in modulating processes associated with aging, disease development and increased risk for diseases. We will explore the association between pyridoxal 5’-phosphate, the active form of vitamin B6 and inflammation by measuring the immunomodulatory compounds in plasma that are produced from pyridoxal 5’-phosphate-dependent reactions in a cohort of elders. Decrease in vitamin B12 status can potentially result in decrease in muscle strength. We will explore the association between vitamin B12 and muscle strength and power, to determine the feasibility of an intervention trial to improve muscle strength. Fortification of cereals with folic acid was adopted by the US and many other countries to reduce neural tube defects. While natural folate from plants and meat can enter the metabolic pathway directly, folic acid, the synthetic form of the vitamin in supplements and fortified foods, has to be reduced by dihydrofolate reductase prior to entering the metabolic pathway. A 19bp deletion polymorphism in intron 1 of dihydrofolate reductase has been associated with increased risk for cancer in supplement users. We will determine the effect of this 19bp deletion on gene expression and enzyme activity of dihydrofolate reductase and the reactions of folate pathway. Intake of folic acid in excess of the ability of the body to metabolize it has been associated with negative health outcomes. Using a mouse model we will determine the effect of excess intake of folic acid on immune function and response to infections. We will explore the association between the polymorphism in dihydrofolate reductase gene and folic acid intake in modulating the risk for breast cancer using the samples and data from the PLCO cohort.

Progress Report
This report documents research conducted under 1 project in a Non-Assistance Cooperative Agreement between ARS and TUFTS UNIVERSITY. Additional details for the research are associated with project 8050-51000-093-01S, One Carbon Nutrients and Metabolsim. Pyridoxal 5’phosphate (PLP), the bioactive form of vitamin B6, is a cofactor for enzymes in the kynurenine pathway of tryptophan degradation that produce immunomodulatory compounds. Early studies suggest that one of these compounds, xanthurenic acid (XA), has diabetogenic effects by chelating with insulin and reducing its hormonal activity. We are in the process of determining the relation of PLP, XA, insulin resistance and diabetes in a population of Boston-area elderly over 60 years of age. Our preliminary analysis of the data shows that higher plasma XA was associated with higher insulin resistance and higher odds of having diabetes. Higher ratio of XA to its precursor, hydroxykynurenine, is associated with increased insulin resistance. Our data suggests that PLP is involved in development of diabetes via xanthurenic acid production in the kynurenine pathway. We have previously shown that low vitamin B12 status can result in cognitive impairment and neuropathy which is exacerbated by high folate status. The 776 C>G genetic variation of the vitamin B-12 transport protein transcobalamin II (TC2) gene, which substitutes proline at position 259 of the protein to arginine, is associated with lower holo-transcobalamin (transcobalamin protein bound to B12) concentration in plasma and can reduce the availability of vitamin B12 to tissues. We determined the effect of the genetic variation in TC2 (resulting in CC, CG and GG genotypes) and its interaction with folate intake on cognitive function and neuropathy in a population of vitamin B12 replete homebound elders aged 60 years and older. The variant GG genotype was associated with lower plasma vitamin B12 when compared to CC genotype. Scores for Mini Mental State Examination which tests cognitive function were slightly lower in GG genotypes compared to CG genotypes but not scores of memory, executive function, or attention. Odds for neuropathy were 3 fold higher in individuals with the variant TC2 when compared to those who did not have the variation. These odds were increased to 5.4 fold when folate intake was above 800mcg dietary folate equivalent, but not when intake was below 800 mcg folate. Our study shows that genetic variation in TC2 increased odds for neuropathy and this association was modified by high folate intake even in vitamin B12 sufficient elders. Folic acid, the synthetic form of folate used in supplements and fortified foods, has to be reduced by dihydrofolate reductase before it can enter the metabolic pathway. A 19bp deletion polymorphism (19bpdel) in intron 1 of DHFR is associated with increased risk for breast cancer in women who use multivitamin supplements, and retinoblastoma in children if their mothers used folic acid supplements during pregnancy. This polymorphism is also associated with reduced incorporation of folic acid in to tissues, suggesting that the polymorphism may affect DHFR expression or activity. We sought to determine the effect of the 19bpdel polymorphism on DHFR expression in women aged 21 to 45. 130 women were recruited for the study and 117 were eligible to participate in the study. Genotype analysis showed that there were 30 DHFR19bpdel homozygotes, 55 heterozygotes and 32 without the polymorphism in this cohort. Gene expression analysis was conducted in peripheral mononuclear blood cells. In DHFR 19bpdel homozygotes, the DHFR transcript was 1.5 fold higher when compared to those without the polymorphism, and 1.4 fold higher when compared to the heterozygotes. Differences in DHFR protein could not be determined in the unstimulated blood cells under the conditions of the assay. We are currently conducting analyses on cells that have been stimulated to grow in culture.

Accomplishments
1. High plasma folate concentration is negatively associated with telomere length. Shortening of telomeres, the protective structures at the ends of human chromosomes, is associated with age-related diseases. Telomere length is influenced by DNA integrity and by methyl group chemical modification of DNA and DNA associated proteins called histones, some of which is caused by folate. ARS funded researchers at the Jean Mayer USDA Human Nutrition Research Center on Aging in Boston, Massachusetts, in collaboration with colleagues at Framingham Heart Study, determined the relationship between telomere length of white blood cells and folate nutritional status in participants of Framingham Offspring Study. Samples for this study had been collected before and after folic acid fortification of cereal grains mandated by FDA. Contrary to expectation, high folate status was associated with shorter telomeres, as was multivitamin use. While adequate folate nutrition is necessary for maintaining telomere health, high plasma folate status possibly resulting from excess folic acid intake may interfere with the role of folate in maintaining telomere integrity.

2. Arsenic is associated with reduced effect of folic acid in neural tube defect prevention. Exposure to arsenic is a public health concern in areas where drinking water is contaminated with naturally occurring arsenic, or in areas where residual lead arsenate from pesticides is still present in soil. Arsenic detoxification potentially involves the addition of methyl chemical groups to arsenic, and adequate folate nutrition may reduce toxicity due to arsenic exposure. ARS funded researchers at the Jean Mayer USDA Human Nutrition Research Center on Aging in Boston, Massachusetts, in collaboration with colleagues at Boston Children’s Hospital, Harvard School of Public Health and Dhaka Community Hospital in Bangladesh, determined the whether folate nutritional status of mothers exposed to naturally occurring arsenic in drinking water was associated with neural tube defects of their children in Bangladesh. Periconceptional folic acid use protected against neural tube defect due to arsenic in drinking water, but this protective effect declined when drinking water arsenic concentrations increased. The results of this study suggest that folic acid supplementation alone is not sufficient in preventing neural tube defects due to environmental arsenic exposure.

3. Association between vitamin B12, albuminuria and reduced kidney function. Variants in CUBN, the gene encoding cubilin, a proximal tubular transport protein, have been associated with vitamin B12 deficiency. Cubilin also acts to reabsorb the majority of filtered albumin from the urine and variants in CUBN are associated with albuminuria, which is often a symptom of kidney disease. ARS funded researchers at the Jean Mayer USDA Human Nutrition Research Center on Aging in Boston, Massachusetts, in collaboration with scientists from Framingham Heart Study and University of Alabama at Birmingham, tested the association between vitamin B12 and prevalent albuminuria and reduced kidney function in participants from the Framingham Heart Study and National Health and Nutritional Examination Survey 2003-2004. In Framingham cohort, elevated vitamin B12 was associated with albuminuria, but this was not seen in the NHANES. Elevated vitamin B12 was associated with reduced kidney function in individuals with high baseline homocysteine levels, which is a functional marker for vitamin B12. The combination of elevated homocysteine along with increased vitamin B12 suggests the possibility of a resistance to the usual effects of B12 in these individuals.