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.
