Reduced folate

Methotrexate (MTX, chemical structure shown in Fig. 1.) competitively inhibits the dehyrofolate reductase, an enzyme that plays an essential role in purine synthesis. The dehydrofolate reductase regenerates reduced folates when thymidine monophosphate is formed from deoxyuridine monophosphate. Without reduced folates cells are unable to synthesize thymine. Administration of N-5 tetrahydrofolate or N-5 formyl-tetrahydrofolate (folinic acid) can bypass this block and rescue cells from methotrexate activity by serving as antidote. 

Fluorouracil (5-fluorouracil, 5-FU, Fig. 5) represents an early example of rational drag design in that it originated from the observation that tumor cells, especially from gut, incorporate radiolabeled uracil more efficiently into DNA than normal cells. 5-FU is a fluorinated pyrimidine analog that must be activated metabolically. In the cells 5-FU is converted to 5-fluoro-2>deoxyuridine-monophosphate (FdUMP). This metabolite inhibits thymidilate synthase which catalyses the conversion of uridylate (dUMP) to thymidilate (dTMP) whereby methylenetetrahydrofo-late plays the role of the carbon-donating cofactor. The reduced folate cofactor occupies an allosteric site of... 

Folates carry one-carbon groups in transfer reactions required for purine and thymidylic acid synthesis. Dihydrofolate reductase is the enzyme responsible for supplying reduced folates intracellularly for thymidylate and purine synthesis. 

Eudy, J. D., et al. Identification and characterization of the human and mouse SLC19A3 gene a novel member of the reduced folate family of micronutrient transporter genes. Mol. Genet. Metab. 2000, 73, 581-590. 

Quantitative molecular-property relationship Segregated-flow model Reduced folate carrier protein... 

The answer is c. (Katzung, pp 608-609, 932-9.13.) Methotrexate is classified as an anti metabolite with therapeutic uses in cancer chemotherapy and as an immunosuppressive agent indicated in the treatment of severe active classical rheumatoid arthritis. Leucovorin is related to methotrexate in that it is an antagonist of its actions. It can supply a source of reduced folate for the methylation reactions that are prevented by methotrexate. 

In humans and rats, early investigations showed that large doses of folic acid resulted in increased excretion of a substance that stimulated the growth of P. cerevisiae and that was presumed to be citrovorum factor.45 This response is now also associated with other reduced folates. 

A later paper65 reported a fluorescence maximum for leucovorin at 365 nm when excited at 314 nm in a pH 7 solution the concentration was 5 x 10-5 M. Variation between these data and other values was attributed to sample impurity, pH of solution, and quenching. The authors made an attempt to correlate structure and fluorescence of reduced folates. Similarity between tested compounds and jj-aminobenzoyl-glutamate lead them to conclude that this portion of the molecule is responsible for maxima at 360-425 nm when excited at 300-320 nm. They suggested that intensity differences may arise from various substitutions on the tetra-hydropteridine moiety. 

Although this method was designed for folates in general, differential microbiological testing could give an indication of reduced folate concentration. 

Fig. 14.1 Cellular pathway of methotrexate. ABCBl, ABCCl-4, ABC transporters ADA, adenosine deaminase ADP, adenosine diphosphate AICAR, aminoimidazole carboxamide ribonucleotide AMP, adenosine monophosphate ATIC, AICAR transformylase ATP, adenosine triphosphate SjlO-CH -THF, 5,10-methylene tetrahydrofolate 5-CHj-THF, 5-methyl tetrahydro-folate DHFR, dihydrofolate reductase dTMP, deoxythymidine monophosphate dUMP, deoxy-uridine monophosphate FAICAR, 10-formyl AICAR FH, dihydrofolate FPGS, folylpolyglutamyl synthase GGH, y-glutamyl hydrolase IMP, inosine monophosphate MTHFR, methylene tetrahydrofolate reductase MTR, methyl tetrahydrofolate reductase MTX-PG, methotrexate polyglutamate RFCl, reduced folate carrier 1 TYMS, thymidylate synthase. Italicized genes have been targets of pharmacogenetic analyses in studies published so far. (Reproduced from ref. 73 by permission of John Wiley and Sons Inc.)...

Rothem, L., Aronheim, A., and Assaraf, Y.G. (2003) Alterations in the expression of transcription factors and the reduced folate carrier as a novel mechanism of antifolate resistance in human leukemia cells. Journal of Biological Chemistry. 278, 8935-8941. 

Whetstine, J.R., Witt, T.L., and Matherly, L.H. (2002) The human reduced folate carrier gene is regulated by the AP2 and spl transcription factor families and a functional 61-base pair polymorphism. Journal of Biological Chemistry. 277, 43873-43880. 

Dervieux, T., Eurst, D., Lein, D. O., et al. (2004) Polyglutamation of methotrexate with common polymorphisms in reduced folate carrier, aminoimidazole carboxamide ribonucleotide transformylase, and thymidylate synthase are associated with methotrexate effects in rheumatoid arthritis. Arthritis and Rheumatism. 50, 2766-2774. 

Following oral administration leucovorin is rapidly absorbed and expands the serum pool of reduced folates. Oral absorption of leucovorin is saturable at doses greater than 25 mg. The apparent bioavailability of leucovorin was 97% for 25 mg, 75% for 50 mg, and 37% for 100 mg. 

Reduced folate Reduction of serum or red cell folate has been reported over long-term administration of cholestyramine. Consider supplementation with folic acid. 

The DNA-mediated effects of fluropyrimidines can be modulated by a number of agents, such as leucovorin (LV), levamisole, and interferon-alpha (IFN-alpha). LV prolongs TS inhibition by increasing the availability of the reduced folate cofactor necessary for formation of the inactive TS-FdUMP complex (21) (Fig. 4). Studies show alpha-interferon can potentiate 5-FU-mediated cyotoxicity, but the mechanisms are not yet defined (22,23). Another approach to modulate the activity of fluoropyrimidines is the use of the nucleoside transport inhibitor dipyridamole. Dipyridamole probably permits... 

Natnral folate, introduced through the diet, are mostly reduced folates, i.e., derivatives of tetra-hydrofolate (THE), such as 5-methyl-THF (5-MTHF), 5-formyl-THF, and 5,10-methylene-THF, and exist mainly as pteroylpolyglntamates, with np to nine additional glutamate molecules attached to the pteridine ring. 

Folate supplementation of food in the United States is expected to reduce folate-associated birth defects by up to 70%. 

The adverse effects of methotrexate include gastrointestinal complaints, bone marrow suppression, alopecia and nephrotoxicity. The toxic effects of methotrexate may be terminated by administering the fully reduced folate coenzyme leucovorin (folinic acid). Leucovorin rescue permits the administration of high doses of methotrexate, for example in situations where partially resistance has occurred or to obtain cytotoxic concentrations of methotrexate in the CNS. 

The carbon-donating cofactor for this reaction is N, N methylenetetrahydrofolate, which is converted to dihydrofolate. The reduced folate cofactor occupies an allosteric site on thymidylate synthetase, which allows for the covalent binding of 5-FdUMP to the active site of the enzyme. 

Thymidylate Synthase Gene Reduced Folate Carrier (RFC) Gene Discussion References... 

The reduced folate carrier is the major transporter of methotrexate into cells. The RFC gene is comprised of 40,719 base pairs, has 12 exons, and is located on 21q22.3. The primary polymorphisms studied to date is the RFCl G80A variant, which is associated with a decrease in MTX transport into cells. Rady et al. noted similar rates of variation in several ethnic groups variant homozygosity rates were 28.7% in individuals of Ashkenazi-Jewish decent, 20.8% in African-American, 29% in Caucasians, and 26% in Hispanics (23). 

Shimasaki N, Mori T, Samejima H et al. Effects of methylenetetrahydrofolate reductase and reduced folate earrier 1 polymorphisms on high-dose methotrexate-induced toxicities in children with acute lymphoblastic leukemia or lymphoma. JPediatr Hematol Oncol 2006 28 64-68. 

Rady PL, Szucs S, Matalon RK et al. Genetic polymorphism (G80A) of reduced folate carrier gene in ethnic populations. Mol Genet Metab 2001 73 285-286. 

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