Vitamin B6 Effects on One-Carbon Metabolism

描述（由申请人提供）：维生素B6在人体一碳（1C）代谢中发挥关键作用，通过磷酸吡哆醛（PLP）作为关键反应的辅酶的基本功能，包括由线粒体甘氨酸裂解系统（GCS）、线粒体和细胞质丝氨酸羟甲基转移酶（SHMT）异构体催化的反应，以及同型半胱氨酸分解代谢的转硫途径的酶。本申请请求支持延长基金DK072398（维生素B6对单碳代谢的影响）的Aim 1，该项目涉及人体甘氨酸代谢及其维生素B6依赖性的定量表征。我们最近完成了最初计划的Aim 1方案，完全符合我们的资助资助。该方案包括以[1,2- 13c2]甘氨酸作为主要示踪剂的稳定同位素输注，以研究甘氨酸动力学、甘氨酸到丝氨酸的相互转化、甘氨酸裂解动力学以及甘氨酸作为碳供体在单碳代谢中的作用（在控制维生素B6消耗之前和之后进行研究）。双标记甘氨酸示踪剂在本研究中具有许多属性。该方法的一个关键方面是测定呼吸样本中13CO2的演变，指示甘氨酸脱羧过程（主要通过GCS）。然而，我们不能保证13CO2的产生主要来自甘氨酸13c -羧基，而来自C-2位置的13CO2的产生（例如，通过10-甲酰基四氢叶酸脱氢酶）可以忽略不计。澄清这一问题的建议输注方案将与我们已完成的Aim 1研究中使用的相同，除了输注的标记甘氨酸将是[1- 13c]甘氨酸（即仅在羧基上标记）。受试者（n=6，维生素B6充足）将从完成Aim 1研究的同一个人中招募。在这个过程中，呼出13CO2的产生将具体反映甘氨酸脱羧。将比较双标记甘氨酸（先前的Aim 1研究）与新研究中使用的羧基标记甘氨酸在每个受试者中产生的13CO2。这些示踪剂的高原13CO2产量与的13CO2产量曲线下的面积之间的差异将反映Aim 1中甘氨酸C-2碳次生13CO2的生产程度。因此，我们将能够充分说明甘氨酸脱羧的程度，而不是通过10-甲酰基四氢叶酸脱氢酶等反应氧化甘氨酸C-2衍生的一碳单位。总的来说，这些研究将产生关于甘氨酸衍生碳进入人体单碳代谢的来源和处置的新信息。因此，这些信息将增强我们评估慢性疾病与这些途径的营养/代谢紊乱之间联系的能力。公共卫生相关性：单碳代谢是人体合成核苷酸（RNA和DNA的组成部分）和一碳单位（包括在代谢和遗传调控中至关重要的甲基）的生化过程的集合。包括血栓、中风和某些形式的心脏病在内的慢性疾病的风险与单碳代谢的营养或基因变化有关。拟议的研究将扩展我们以前的研究，所有这些研究的主要目的都是提高我们对这些代谢过程的速率及其对营养和遗传变量的反应的理解。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: 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.