METABOLIC ASPECTS OF COBALAMIN AND FOLATE PATHWAYS

这项研究计划的主要目标是继续发展 钴胺(CB1)类似物，对CB1和叶酸有抑制作用 新陈代谢，并确定代谢变化和改善 CB1和叶酸缺乏的诊断。 初步研究已证实CB1依赖的抑制作用 某些CB1类似物对大鼠的酶活性。调查 该提案中概述的内容包括合成和纯化CB1类似物 具有更强的抑制作用，并优化了 抑制力。 CB1类似物抑制CB1依赖代谢的机制 将会被调查。将确定是否生产了CB1类似物 细胞内CB1的辅酶形式，无论它们是否与 L-甲基丙二酰辅酶A变位酶和蛋氨酸合成酶的类似物 以其各自的辅酶形式直接抑制这两种酶，并且 它们是否会耗尽CB1的细胞。 具有最强抑制作用的CB1类似物将用于 建立一种更快速、更方便的神经系统疾病动物模型 通过给果蝠注射CB1来缓解CB1缺乏。 CB1类似物将被测试为抑制人类造血细胞生长 细胞系。具有抑制作用的类似物将被测试 人异基因移植对裸鼠化疗效果的影响 造血细胞肿瘤。 使用灵敏和特异的气相色谱/质谱学方法 分析，血清中甲基丙二酸和琥珀酸的水平， 低血压病患者将检测同型半胱氨酸、蛋氨酸和半胱氨酸 或接近低水平的血清CB1和叶酸，这些水平将是 巨幼细胞性贫血的临床表现与神经学的关系 异常以提高对CB1和叶酸的诊断 缺乏症。大剂量一氧化二氮麻醉病人 甲氨蝶呤治疗也将被分析，以更好地定义代谢 在这些临床情况下的异常。将使用类似的方法 定量检测CB1患者血清β-亮氨酸和亮氨酸水平 缺乏症和正常受试者。

The major objectives of this research proposal are to continue to develop cobalamin (Cb1) analogues that have inhibitory effects on Cb1 and folate metabolism, and to define the metabolic changes of and improve the diagnosis of Cb1 and folate deficiency. Preliminary studies have demonstrated the inhibition of Cb1-dependent enzyme activity in rats by certain Cb1 analogues. The investigations outlined in this proposal include synthesizing and purifying Cb1 analogues with more potent inhibitory effects, and optimizing the conditions for the inhibition. The mechanisms of inhibition of Cb1 analogues on Cb1-dependent metabolism will be investigated. It will be determined whether Cb1 analogues are made into the coenzyme forms of Cb1 intracellularly, whether they bind to L-methylmalonyl-CoA mutase and methionine synthetase, whether the analogues in their respective coenzyme forms directly inhibit the two enzymes, and whether they deplete cells of Cb1. The Cb1 analogues with the most potent inhibitory effects will be used to develop a more rapid and convenient animal model of the neurologic disease of Cb1 deficiency by administering them to fruit bats. Cb1 analogues will be tested for growth suppression of human hematopoietic cell lines. Analogues with inhibitory effects will be tested for chemotherapeutic effectiveness in nude mice with heterotransplants of human hematopoietic tumors. Using sensitive and specific gas chromatographic/mass spectrometric methods of analysis, serum levels of methylmalonic and succininc acids, homocysteine, methionine and cysteine will be measured in patients with low or borderline low serum levels of Cb1 and folate, and these levels will be correlated with clinical findings of megaloblastic anemia and neurologic abnormalities in order to improve the diagnosis of Cb1 and folate deficiency. Patients receiving nitrous oxide anesthesia and high dose methotrexate therapy will also be analyzed to better define the metabolic abnormalities in these clinical situations. Similar methods will be used to quantitate serum levels of Beta-leucine and leucine in patients with Cb1 deficiency and in normal subjects.