Vitamin B6 plays a critical role in human one-carbon (1C) metabolism through the essential function of pyridoxal phosphate (PLP) as coenzyme for key reactions including those catalyzed by the mitochondrial glycine cleavage system (GCS), mitochondrial and cytosolic serine hydroxymethyltransferase (SHMT) isoforms, and the enzymes of the transsulfuration pathway of homocysteine catabolism. This application requests support to extend Aim 1 of grant DK072398 (Vitamin B6 Effects on One-Carbon Metabolism), which addresses the quantitative characterization of human glycine metabolism and its vitamin B6-dependence. We recently completed the originally planned Aim 1 protocol exactly as described in our funded grant. This protocol involved a stable isotopic infusion with [1,2-13C2]glycine as the primary tracer to investigate glycine kinetics, glycine-to-serine interconversion, glycine cleavage kinetics, and the role of glycine as a carbon donor in one-carbon metabolism (examined before and after controlled vitamin B6 depletion). The doubly-labeled glycine tracer has many attributes for this study. A key aspect of the method is the determination of the evolution of 13CO2 in breath samples, indicative of glycine decarboxylation processes (primarily via the GCS). However, we cannot be assured that the generation of 13CO2 is primarily from the glycine13C-carboxyl group and that 13CO2 generation from the C-2 position (e.g., via 10-formyltetrahydrofolate dehydrogenase) is negligible. The proposed infusion protocol to clarify this issue will be identical that used in our completed Aim 1 study with the exception that the labeled glycine infused will be [1-13C]glycine (i.e., labeled only on the carboxyl group). Subjects (n=6, vitamin B6 adequate) will be recruited from the same individuals who completed the Aim 1 study. In this procedure, the generation of breath 13CO2 specifically will reflect glycine decarboxylation. Comparisons will be made of 13CO2 production in each subject between doubly-labeled glycine (previous Aim 1 study) versus carboxyl labeled glycine in the new study using. Any difference between these tracers in plateau 13CO2 production and the areas under the 13CO2 production curves of will reflect the extent of secondary 13CO2 production from the C-2 carbon of glycine in Aim 1. Thus, we will be able to account fully for the extent by which glycine is decarboxylated versus the oxidation of glycine C-2 derived one-carbon units via reactions such as 10-formyltetrahydrofolate dehydrogenase. Overall, these studies will yield novel information regarding the sources and disposition of glycine-derived carbons entering human one-carbon metabolism. This information will, thus, enhance our ability to evaluate linkages between chronic disease and nutritional/metabolic perturbations of these pathways.
One-carbon metabolism is the collection of biochemical processes in which the body synthesizes nucleotides, the building blocks of RNA and DNA, and one-carbon units including methyl groups that are vital in metabolism and genetic regulation. Risk of chronic disease including blood clots, stroke and some forms of heart disease are linked to nutritional or genetic changes in one-carbon metabolism. The proposed research will extend our previous studies, all of which have the major objective of improving our understanding of the rates of these metabolic processes and their response to nutritional and genetic variables. ? ? ?
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