Vitamin C (ascorbic acid) Biochemistry and Molecular Biology

维生素 C（抗坏血酸）生物化学和分子生物学

• 批准号: 10697777
• 负责人: MARK A LEVINE
• 金额: $ 46.86万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10697777
• 关键词: Animals; Ascorbic Acid; Biochemistry; Blood Component Removal; Brain; Cells; Computer software; DNA Methylation; Data; Data Analyses; Depressed mood; Disease; Dose; Drug Kinetics; Epithelial; Excretory function; Filtration; Gene Expression; Genes; Genetic; Genetic Transcription; Growth; Human; Inbred BALB C Mice; Inbreeding; Individual; Intake; Intestinal Absorption; Investigation; Kidney; Knockout Mice; Laboratories; Lymphocyte; Messenger RNA; Molecular Biology; Mus; Neurons; Nutrient; Pathway interactions; Physiological Processes; Plasma; Play; Population; RNA; Recommended Dietary Allowance; Regulation; Single Nucleotide Polymorphism; Testing; Time; Tissues; Urine; Weaning; Whole Blood; Woman; ascorbate; base; human subject; in vivo; monocyte; response; trend

Summary Based on extensive pharmacokinetics data from this laboratory, we have learned that Vitamin C concentrations are tightly controlled in healthy humans as a function of vitamin C dose. Three physiologic processes appear to be responsible: intestinal absorption, tissue transport, and renal filtration/reabsorption. In particular, our pharmacokinetics data indicate that the renal threshold of excretion plays a key role in the observed tight control of vitamin C concentrations in plasma and tissues. We hypothesized that Slc23a1, the vitamin C epithelial transporter expressed in kidney, is responsible for tight control. To test this we created a mouse null for the Slc23a1 gene. Inbred C57bL/6J wild type, Slc23a1+/- and Slc23a1-/- mice were generated on a Balb/c background. After weaning, growth rates of wild type, Slc23a1+/- and Slc23a1-/- mice were indistinguishable. Urine vitamin C fractional excretion was 7-10 fold higher in Slc23a1-/- mice compared to wildtype littermates. Corresponding plasma ascorbic acid concentrations in slc23a1-/- mice were 50-70% lower than in wild type littermates. In slc23a1-/- mice, neuronal tissue ascorbate concentrations levels were unaffected by the depressed plasma ascorbate levels, whereas all other tissues trended downwards. These data show that Slc23a1 is responsible for tight control of vitamin C, via regulation of renal threshold of excretion. The asymmetric response of tissues to decreased plasma vitamin C may represent redistribution of vitamin C to protect brain function. Active investigation is underway to describe the consequences of the vitamin C renal leak in these animals. Humans with Slc23a1 single nucleotide polymorphisms that decrease activity of the renal vitamin C transporter might also have altered vitamin C pharmacokinetics due to a renal leak, and therefore require additional vitamin C intake. These findings have unique implications for recommended dietary allowances, because they may need to account for individual genetic differences. Vitamin C is accumulated in human lymphocytes and monocytes, but its function is unknown. We hypothesized that vitamin C might regulate gene transcription in these cells. To investigate, we studied lymphocyte-enriched and monocyte-enriched cell populations isolated from whole blood by in vivo apheresis from 11 healthy women who were 19-27 years old. Cells were isolated from the same women at steady state at each of the following daily vitamin C doses: 30mg; 60 mg; 100 mg; and 1000 or 2500 mg. Comparisons to determine significant changes in gene expression were performed using mRNA from the same subjects at different doses, with RNA obtained at 30 mg as baseline. Gene expression was analyzed using Affymatrix arrays, statistical and pathway software (GeneSpring, Partek, GeneGo), and confirmed by quantitative real time PCR. To confirm and further explore observed pathway changes, DNA methylation was analyzed as a function of vitamin C dose. Extensive data analyses are in progress. This approach provides a new means to characterize changes in gene expression as a function of varying nutrient concentration in the same human subject, for any nutrient.

概括 根据该实验室的大量药代动力学数据，我们了解到，健康人体中的维生素 C 浓度受到严格控制，与维生素 C 剂量有关。三个生理过程似乎是造成这种情况的原因：肠道吸收、组织运输和肾脏过滤/重吸收。特别是，我们的药代动力学数据表明，肾排泄阈值在观察到的血浆和组织中维生素 C 浓度的严格控制中起着关键作用。我们假设 Slc23a1（肾脏中表达的维生素 C 上皮转运蛋白）负责严格控制。为了测试这一点，我们创建了 Slc23a1 基因无效的小鼠。近交 C57bL/6J 野生型、Slc23a1+/- 和 Slc23a1-/- 小鼠在 Balb/c 背景上生成。断奶后，野生型、Slc23a1+/- 和 Slc23a1-/- 小鼠的生长速度没有区别。与野生型同窝小鼠相比，Slc23a1-/- 小鼠的尿液维生素 C 排泄分数高出 7-10 倍。 slc23a1-/- 小鼠中相应的血浆抗坏血酸浓度比野生型同窝小鼠低 50-70%。在 slc23a1-/- 小鼠中，神经元组织的抗坏血酸浓度水平不受血浆抗坏血酸水平降低的影响，而所有其他组织均呈下降趋势。这些数据表明，Slc23a1 通过调节肾排泄阈值负责严格控制维生素 C。组织对血浆维生素 C 减少的不对称反应可能代表维生素 C 的重新分配以保护大脑功能。目前正在进行积极的调查，以描述这些动物体内维生素 C 肾渗漏的后果。具有 Slc23a1 单核苷酸多态性（可降低肾脏维生素 C 转运蛋白活性）的人类也可能因肾漏而改变维生素 C 药代动力学，因此需要额外摄入维生素 C。这些发现对推荐的膳食摄入量具有独特的影响，因为它们可能需要考虑个体遗传差异。 维生素C在人体淋巴细胞和单核细胞中积累，但其功能尚不清楚。我们假设维生素 C 可能调节这些细胞中的基因转录。为了进行调查，我们研究了通过体内单采术从 11 名 19-27 岁健康女性的全血中分离出的富含淋巴细胞和富含单核细胞的细胞群。在以下每日维生素 C 剂量下，从同一女性中分离出处于稳定状态的细胞：30mg； 60毫克； 100毫克；和 1000 或 2500 毫克。使用来自同一受试者不同剂量的 mRNA 进行比较，以确定基因表达的显着变化，以 30 mg 获得的 RNA 作为基线。使用 Affymatrix 阵列、统计和通路软件（GeneSpring、Partek、GeneGo）分析基因表达，并通过定量实时 PCR 进行确认。为了确认并进一步探索观察到的途径变化，对 DNA 甲基化作为维生素 C 剂量的函数进行了分析。广泛的数据分析正在进行中。这种方法提供了一种新方法，可以将基因表达的变化描述为同一人类受试者中任何营养素不同营养素浓度的函数。