A deficiency of folic acid leads to a life-threatening megaloblastic anemia. Vitamin B12 deficiency and exposure to the anesthetic gas, nitrous oxide (N20)- causes a functional folate deficiency by trapping of folate coenzymes, needed for DNA and RNA synthesis, as 5-methyltetrahydrofolate. This trapping leads to megaloblastic anemia and to neuropathy. Folates in the liver cell are partitioned between the cytosol and the mitochondria. Recent studies have shown that short-term N20 inactivation of the B12-dependent enzyme, methionine synthase, results in trapping of hepatic folates as 5-methyltetrahydrofolate and greatly decreased concentrations of other reduced folates only in the cytosol. Mitochondrial folate coenzymes were not affected. Therefore, it is quite evident, not only, that folates are compartmentalized in the cell but that nutritional and maybe other factors which affect folate metabolism may do so differently in these cellular compartments. The long-term objective of this proposal is to delineate the subcellular metabolism of folate coenzymes in liver. The effects of long-term N20 exposure and of methionine on cytosolic and mitochondria folate-requiring enzymes will be studied, as will the effects on folate coenzyme and S-adenosylmethionine levels (assayed by high-pressure liquid chromatography). The effects of folic acid deficiency on cytosolic and mitochondrial folate enzyme activity and enzyme protein levels (determined by enzyme-linked immunoassay) and on folate coenzymes will likewise be determined. Mitochondrial transport of folate coenzymes will be studied. Whether mitochondria synthesize S-adenosylmethionine (needed for DNA and RNA methylation) and/or obtain it by transport from the cytosol is not known and will be investigated. The antifolate cancer drug, methotrexate, is being used in long-term treatment of severe psoriasis and rheumatoid arthritis. One of the side effects is hepatic toxicity. The contribution of mitochondrial enzyme inhibition to this toxicity has not been addressed. The distribution of methotrexate between cytosol and mitochondria will be determined. The presence of methotrexate polyglutamate derivatives in these compartments will be assessed, as will the degree of inhibition of folate-requiring enzymes by methotrexate and its polyglutamate derivatives.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK032189-08
Application #
3230635
Study Section
Nutrition Study Section (NTN)
Project Start
1983-06-01
Project End
1993-05-31
Budget Start
1991-06-01
Budget End
1992-05-31
Support Year
8
Fiscal Year
1991
Total Cost
Indirect Cost
Name
Vanderbilt University Medical Center
Department
Type
Schools of Medicine
DUNS #
004413456
City
Nashville
State
TN
Country
United States
Zip Code
37212
Cai, Songmin; Horne, Donald W (2003) Transport of 5-formyltetrahydrofolate into primary cultured cerebellar granule cells. Brain Res 962:151-8
Cai, Songmin; Horne, Donald W (2003) Transport of 5-formyltetrahydrofolate into primary cultured rat astrocytes. Arch Biochem Biophys 410:161-6
Horne, Donald W (2003) Neither methionine nor nitrous oxide inactivation of methionine synthase affect the concentration of 5,10-methylenetetrahydrofolate in rat liver. J Nutr 133:476-8
Horne, Donald W; Reed, Kathleen A (2003) Uptake of 5-methyltetrahydrofolate into PC-3 human prostate cancer cells is carrier-mediated. J Nutr Biochem 14:473-9
Roje, Sanja; Chan, Sherwin Y; Kaplan, Fatma et al. (2002) Metabolic engineering in yeast demonstrates that S-adenosylmethionine controls flux through the methylenetetrahydrofolate reductase reaction in vivo. J Biol Chem 277:4056-61
Horne, Donald W; Reed, Kathleen A (2002) Transport of methotrexate into LNCaP human prostate cancer cells. Biofactors 16:19-27
Horne, D W (2001) High-performance liquid chromatographic measurement of 5,10-methylenetetrahydrofolate in liver. Anal Biochem 297:154-9
Horne, D W; Reed, K A (2001) Transport of methotrexate into PC-3 human prostate cancer cells. Arch Biochem Biophys 394:39-44
Koury, M J; Horne, D W; Brown, Z A et al. (1997) Apoptosis of late-stage erythroblasts in megaloblastic anemia: association with DNA damage and macrocyte production. Blood 89:4617-23
Horne, D W; Holloway, R S (1997) Compartmentation of folate metabolism in rat pancreas: nitrous oxide inactivation of methionine synthase leads to accumulation of 5-methyltetrahydrofolate in cytosol. J Nutr 127:1772-5

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