METABOLIC ASPECTS OF COBALAMIN AND FOLATE PATHWAYS

这项研究计划的主要目标是继续发展 对Cb 1和叶酸具有抑制作用的钴胺素（Cb 1）类似物 代谢，并定义代谢变化和改善 诊断为Cb 1和叶酸缺乏症。 初步研究表明， 某些Cb 1类似物在大鼠中的酶活性。 调查 本文概述了Cb 1类似物的合成和纯化 具有更强的抑制作用，并优化了 抑制作用 Cb 1类似物抑制Cb 1依赖性代谢的机制 将被调查。 将确定是否制备Cb 1类似物 转化为细胞内Cb 1的辅酶形式，无论它们是否与 L-甲基丙二酰辅酶A和甲硫氨酸合成酶的类似物， 以它们各自的辅酶形式直接抑制这两种酶， 是否会耗尽细胞中的Cb 1 具有最强抑制作用的Cb 1类似物将用于 开发一种更快速、更方便的神经系统疾病动物模型， 通过给果蝠服用这些药物来治疗Cb 1缺乏症。 将测试Cb 1类似物对人造血细胞的生长抑制。 细胞系 将测试具有抑制作用的类似物， 裸鼠移植瘤的化疗效果 造血系统肿瘤 使用灵敏和特异的气相色谱/质谱法 分析，甲基丙二酸和琥珀酸的血清水平， 高半胱氨酸、甲硫氨酸和半胱氨酸将在低血糖患者中测量。 或临界低血清水平的Cb 1和叶酸，这些水平将是 与巨幼细胞性贫血和神经系统疾病的临床表现相关 以提高Cb 1和叶酸的诊断 缺陷 接受氧化亚氮麻醉和高剂量的患者 甲氨蝶呤治疗也将进行分析，以更好地确定代谢 这些临床情况的异常。 将使用类似的方法 定量Cb 1患者的血清β-亮氨酸和亮氨酸水平 缺乏和正常受试者。

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.