One-carbon (Cl) metabolism consists of the generation of carbon units for use in cellular processes including DNA synthesis, regeneration of methionine (Met) from homocysteine (Hcy), and methylation of many biological compounds. Conditions that impair one-carbon metabolism (e.g. folate deficiency) are associated with elevation in plasma Hcy and increased risk of vascular disease, certain cancers, and neural tube defects. A common mutation of methylene-tetrahydrofolate reductase (MTHFR), known as the """"""""thermolabile"""""""" or C677T mutant, has been associated with elevations in plasma I-Icy (especially in low folate status), lower plasma folate, altered distribution of erythrocyte folate, potentially increased risk of vascular disease, and decreased risk of colon cancer. The in vivo metabolic effects of the C677T mutation have not been determined directly. Our overall hypothesis is that the rate of acquisition and generation of methyl groups from serine (primary source of C1 units) is reduced in individuals homozygous for the C677T mutation, and that the genotypic effect is greatest when folate nutriture is inadequate. We also hypothesize that the rate of folate-dependent synthesis of nucleotides (purines and thymidylate) will be reduced in folate deficiency but may be enhanced by the C677T mutation. The proposed studies will determine nutritional and genetic dependence of the flow of Cl units from serine (Ser) to Met and from Ser to nucleotides. This protocol also will allow measurement of the transsulfuration pathway of Hcy catabolism important in disposal of excess Hcy.
Specific aims. To determine: (a) The kinetics by which Ser serves as a donor of Cl units for methyl group synthesis and nucleotide synthesis and the possible degree of impairment caused by the C677T mutation and/or low folate status. (b) The influence of the C677T mutation and folate status on cellular Cl status as reflected by the distribution of folate species in erythrocytes. (C) The influence of the C677T mutation and folate status on homocysteine catabolism. (d) The relative contributions of cytosolic and mitochondrial metabolism in the generation of Cl units for synthesis of methyl groups and nucleotides. (e) The significance of mitochondrial glycine cleavage in generation of Cl units. Protocol: In the main protocol, healthy adequately nourished human subjects (20-30 yr) will be classified by MTHFR genotype, (homozygous control and homozygous mutant). Subjects will be given infusions with 13C-serine as primary precursor initially and following 8-wk dietary depletion of 120 ugld folate to evaluate the effect of nutritional and genotypic effects on Cl kinetics. Two variations of this study will be conducted to determine the relative roles of mitochondrial and cytosolic routes of Cl generation from serine and the role of the mitochondrial glycine cleavage pathway. In total, these studies will yield new functional data regarding the effects of folate deficiency, and the influence of common polymorphism of MTHFR.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK056274-02
Application #
6489735
Study Section
Nutrition Study Section (NTN)
Program Officer
May, Michael K
Project Start
2001-04-01
Project End
2004-12-31
Budget Start
2002-01-01
Budget End
2002-12-31
Support Year
2
Fiscal Year
2002
Total Cost
$301,971
Indirect Cost
Name
University of Florida
Department
Nutrition
Type
Schools of Earth Sciences/Natur
DUNS #
073130411
City
Gainesville
State
FL
Country
United States
Zip Code
32611
Quinlivan, Eoin P; Gregory 3rd, Jesse F (2008) DNA digestion to deoxyribonucleoside: a simplified one-step procedure. Anal Biochem 373:383-5
Quinlivan, Eoin P; Gregory 3rd, Jesse F (2007) Reassessing folic acid consumption patterns in the United States (1999 2004): potential effect on neural tube defects and overexposure to folate. Am J Clin Nutr 86:1773-9
Davis, Steven R; Quinlivan, Eoin P; Stacpoole, Peter W et al. (2006) Plasma glutathione and cystathionine concentrations are elevated but cysteine flux is unchanged by dietary vitamin B-6 restriction in young men and women. J Nutr 136:373-8
Lima, Carolina P; Davis, Steven R; Mackey, Amy D et al. (2006) Vitamin B-6 deficiency suppresses the hepatic transsulfuration pathway but increases glutathione concentration in rats fed AIN-76A or AIN-93G diets. J Nutr 136:2141-7
Quinlivan, Eoin P; Hanson, Andrew D; Gregory, Jesse F (2006) The analysis of folate and its metabolic precursors in biological samples. Anal Biochem 348:163-84
Davis, Steven R; Quinlivan, Eoin P; Shelnutt, Karla P et al. (2005) Homocysteine synthesis is elevated but total remethylation is unchanged by the methylenetetrahydrofolate reductase 677C->T polymorphism and by dietary folate restriction in young women. J Nutr 135:1045-50
Davis, Steven R; Scheer, Jennifer B; Quinlivan, Eoin P et al. (2005) Dietary vitamin B-6 restriction does not alter rates of homocysteine remethylation or synthesis in healthy young women and men. Am J Clin Nutr 81:648-55
Quinlivan, Eoin P; Davis, Steven R; Shelnutt, Karla P et al. (2005) Methylenetetrahydrofolate reductase 677C->T polymorphism and folate status affect one-carbon incorporation into human DNA deoxynucleosides. J Nutr 135:389-96
Davis, Steven R; Quinlivan, Eoin P; Shelnutt, Karla P et al. (2005) The methylenetetrahydrofolate reductase 677C->T polymorphism and dietary folate restriction affect plasma one-carbon metabolites and red blood cell folate concentrations and distribution in women. J Nutr 135:1040-4
Davis, Steven R; Stacpoole, Peter W; Williamson, Jerry et al. (2004) Tracer-derived total and folate-dependent homocysteine remethylation and synthesis rates in humans indicate that serine is the main one-carbon donor. Am J Physiol Endocrinol Metab 286:E272-9

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