10-formyltetrahydrofolate and 5-10-methylenetetrahydrofolic-acid

Studies have shown that conversion of leucovorin to the metabolite 5,10-methylenetetrahydrofolate (5,10-CH2FH4) is responsible for enhancement of the antitumor effects of fluorouracil given in combination with leucovorin, but the biochemical basis of this conversion in humans is not fully understood. To determine a possible sequence of metabolic steps, we studied the pharmacokinetics of leucovorin and its reduced folate metabolites in plasma in healthy volunteers. Groups of five subjects were given two equal doses of 10, 25, 125, 250, or 500 mg/m2 leucovorin, one orally and one intravenously at a 30-day interval. A sensitive radioenzymatic method that we developed previously was used to measure plasma concentrations of [S]5-formyltetrahydrofolate, 10-formyltetrahydrofolate (10-CHOFH4), 5-methyltetrahydrofolate (5-CH3FH4), and the combined 5,10-CH2FH4 plus tetrahydrofolate (FH4) pools. Intravenous administration of leucovorin resulted in dose-dependent accumulation of 5,10-CH2FH4 + FH4 exceeding 2 microM at peak levels. After oral and intravenous administration, 10-CHOFH4 and 5,10-CH2FH4 + FH4 exhibited peak levels earlier and were eliminated more rapidly than 5-CH3FH4. Accumulation of all metabolites after intravenous administration was linearly dose dependent, while oral administration appeared to result in saturation. We propose that the host activation of leucovorin suggested by these findings could be responsible for elevation of intratumor 5,10-CH2FH4 levels, thus enhancing the antitumor effects of fluorouracil. These results also suggest that 10-CHOFH4, 5,10-CH2FH4, and FH4 are intermediate metabolites and that 5-CH3FH4 is the terminal metabolite. In addition, our results indicate that attainment of high plasma levels of the metabolites active in modulation of the therapeutic effects of fluorouracil is best achieved through intravenous administration of high doses of leucovorin. Our future studies will address the proposed sequential conversion pathway and, thus, the mechanism by which pharmacologically relevant reduced folates accumulate in plasma after leucovorin administration.

Topics: Administration, Oral; Adult; Female; Humans; Injections, Intravenous; Leucovorin; Male; Random Allocation; Tetrahydrofolates; Time Factors

Folate-dependent one-carbon (C1) metabolism is compartmentalized in the mitochondria and cytosol and is a source of critical metabolites for proliferating tumors. Mitochondrial C1 metabolism including serine hydroxymethyltransferase 2 (SHMT2) generates glycine for de novo purine nucleotide and glutathione biosynthesis and is an important source of NADPH, ATP, and formate, which affords C1 units as 10-formyl-tetrahydrofolate and 5,10-methylene-tetrahydrofolate for nucleotide biosynthesis in the cytosol. We previously discovered novel first-in-class multitargeted pyrrolo[3,2-

Topics: Antineoplastic Agents; Cell Line, Tumor; Cell Membrane; Cytosol; Drug Screening Assays, Antitumor; Gene Knockout Techniques; Glutathione; Glycine Hydroxymethyltransferase; Humans; Leucovorin; Mitochondria; Pancreatic Neoplasms; Purine Nucleotides; Pyrimidines; Pyrroles; Reactive Oxygen Species; Serine; Tetrahydrofolates

ATP is the dominant energy source in animals for mechanical and electrical work (for example, muscle contraction or neuronal firing). For chemical work, there is an equally important role for NADPH, which powers redox defence and reductive biosynthesis. The most direct route to produce NADPH from glucose is the oxidative pentose phosphate pathway, with malic enzyme sometimes also important. Although the relative contribution of glycolysis and oxidative phosphorylation to ATP production has been extensively analysed, similar analysis of NADPH metabolism has been lacking. Here we demonstrate the ability to directly track, by liquid chromatography-mass spectrometry, the passage of deuterium from labelled substrates into NADPH, and combine this approach with carbon labelling and mathematical modelling to measure NADPH fluxes. In proliferating cells, the largest contributor to cytosolic NADPH is the oxidative pentose phosphate pathway. Surprisingly, a nearly comparable contribution comes from serine-driven one-carbon metabolism, in which oxidation of methylene tetrahydrofolate to 10-formyl-tetrahydrofolate is coupled to reduction of NADP(+) to NADPH. Moreover, tracing of mitochondrial one-carbon metabolism revealed complete oxidation of 10-formyl-tetrahydrofolate to make NADPH. As folate metabolism has not previously been considered an NADPH producer, confirmation of its functional significance was undertaken through knockdown of methylenetetrahydrofolate dehydrogenase (MTHFD) genes. Depletion of either the cytosolic or mitochondrial MTHFD isozyme resulted in decreased cellular NADPH/NADP(+) and reduced/oxidized glutathione ratios (GSH/GSSG) and increased cell sensitivity to oxidative stress. Thus, although the importance of folate metabolism for proliferating cells has been long recognized and attributed to its function of producing one-carbon units for nucleic acid synthesis, another crucial function of this pathway is generating reducing power.

Topics: Animals; Carbon; Cell Line; Cell Line, Tumor; Cytosol; Folic Acid; Glutathione; Glycine; HEK293 Cells; Humans; Isoenzymes; Leucovorin; Methylenetetrahydrofolate Dehydrogenase (NADP); Mice; Mitochondria; NADP; Oxidation-Reduction; Oxidative Stress; Pentose Phosphate Pathway; Serine; Tetrahydrofolates

The kinetic properties of three methylenetetrahydrofolate dehydrogenase-cyclohydrolase (D/C) enzymes (the NADP-dependent bifunctional domain of the human cytoplasmic trifunctional enzyme, the human mitochondrial NAD-dependent bifunctional enzyme, and the NAD(P)-dependent bifunctional enzyme from Photobacterium phosphoreum) were determined in both forward and reverse directions. In the forward direction, the enzymes possess widely different ratios of kcat C/Kcat D, but all channel methenylH4folate produced by the D activity to the C activity with approximately the same efficiency. A deuterium isotope effect is observed with the human NADP-dependent enzyme in both forward and reverse dehydrogenase assays, consistent with hydride transfer being rate limiting for the interconversion of methenyl- and methyleneH4folate. However, no kinetic isotope effect is observed for the overall reverse reaction (formylH4folate to methyleneH4folate). We devised an assay to measure the reverse cyclohydrolase activity independent of the dehydrogenase, and determined that the Kcat (overall reverse) for each enzyme is approximately equal to the Kcat for its reverse cyclohydrolase activity. Therefore, the rate-limiting step in the overall reverse reaction is not hydride transfer by the dehydrogenase, but the production of methenylH4folate catalyzed by the cyclohydrolase. The reverse cyclohydrolase activities of the NADP-dependent D/C and the P. phosphoreum enzymes, but not the mitochondrial NAD-dependent enzyme, can be stimulated 2-fold by the addition of 2',5'-ADP. The results suggest that the cyclohydrolases of the human NADP dependent and P. phosphoreum enzymes are optimized to catalyze the reverse reaction in the presence of bound coenzyme. These results imply that essentially all of the methenylH4folate produced by the cyclohydrolase in the reverse reaction is channeled to the dehydrogenase.

Topics: Adenosine Diphosphate; Aminohydrolases; Bacterial Proteins; Formate-Tetrahydrofolate Ligase; Humans; Kinetics; Leucovorin; Methylenetetrahydrofolate Dehydrogenase (NADP); Mitochondria; Models, Chemical; Multienzyme Complexes; NAD; NADP; Photobacterium; Tetrahydrofolates

The bifunctional dehydrogenase/cyclohydrolase domain of the human NADP-dependent trifunctional methyleneH4folate dehydrogenase/methenylH4folate cyclohydrolase/formylH4folate synthetase (H4folate = tetrahydrofolate) catalyzes two sequential reactions involved in the interconversion of H4folate derivatives. We have established by equilibrium dialysis that a single H4folate-binding site exists per monomer of the dimeric domain and that the presence of nucleotides has two unexpected effects on H4folate substrate binding. Nucleotides containing a 5'-phosphate cause positive cooperativity in the binding of methyleneH4folate but not of 10-formylH4folate, and NADP increases the affinity for 10-formylH4folate by a factor of 25. The results indicate that dinucleotide preferentially binds before 10-formylH4folate in the reverse cyclohydrolase reaction, and this mechanism increases the efficiency of conversion of 10-formylH4folate to methyleneH4folate. We report new kinetic data that are also consistent with a steady-state random mechanism for this enzyme. To assess whether the enzyme functions at equilibrium in vivo, we determined the overall chemical equilibrium constant of Keq = 16 for ([10- formylH4folate][NADPH])/([methyleneH4folate][NADP]). Using this value and reported ratios of free dinucleotides and folate derivatives in vivo, we estimate that the cytosolic dehydrogenase/cyclohydrolase reactions exist near the equilibrium position. However, the NAD-dependent dehydrogenase/cyclohydrolase reactions in mitochondria are far from equilibrium and are poised toward 10-formylH4folate synthesis. The results of the binding and kinetic studies indicate that the bifunctional nature of the methyleneH4folate dehydrogenase/methenylH4folate cyclohdrolase domain is designed to optimize the overall reverse reactions in vivo.

Topics: Aminohydrolases; Binding Sites; Humans; Leucovorin; Methenyltetrahydrofolate Cyclohydrolase; Methylenetetrahydrofolate Dehydrogenase (NADP); Nucleotides; Substrate Specificity; Tetrahydrofolates

A pharmacokinetic study of folic acid and its metabolites was conducted to provide a basis to consider folic acid as a therapeutic alternative to leucovorin. Leucovorin has been used in various folate antagonist rescue regimens and to modulate fluorouracil activity in the treatment of solid tumors. Although leucovorin is typically administered intravenously in fluorouracil modulation therapy, limited oral administration trials have yielded equivalent responses. Because metabolites rather than leucovorin are the predominant circulating species after oral administration, these clinical results indicate that metabolites themselves can be modulating agents. Folic acid could be an attractive alternative to leucovorin provided it effectively elevates the same plasma metabolites. Hence, folic acid at doses of 25 and 125 mg/m2 was administered orally and intravenously to normal volunteers. Plasma folic acid and its reduced folate metabolites were monitored over a 24-hour period by use of a previously developed radioenzymatic method. The metabolites that accumulated--5-methyltetrahydrofolate, 5,10-methylenetetrahydrofolate, tetrahydrofolate, and 10-formyltetrahydrofolate--were the same metabolites that were observed previously after leucovorin administration. Folic acid metabolites accumulated more slowly and persisted longer than leucovorin metabolites, which can be attributed to slower metabolism of the fully oxidized vitamin. Based on these results, it is concluded that folic acid could be an attractive therapeutic alternative to leucovorin for fluorouracil modulation.

Topics: Adult; Female; Folic Acid; Humans; Leucovorin; Male; Reference Values; Tetrahydrofolates

The effect of the inhibition of dihydrofolate reductase by methotrexate on the cellular folates involved in de novo purine and thymidylate biosynthesis has been measured in H35 hepatoma cells grown in 4 microM folic acid or 20 nM folinic acid. The major cellular folate species in cells from medium with folate or folinate is 10-formyltetrahydrofolate (approximately 5 microM), with lesser amounts of 5,10-methylenetetrahydrofolate and tetrahydrofolate. Cultures were exposed to a pulse dose of methotrexate, resulting in the accumulation of nearly exclusively methotrexate polyglutamates (predominantly Glu3, Glu4, and Glu5), or a continuous exposure to the poorly glutamylated analog threo-4-fluoromethotrexate, resulting in 93% intracellular monoglutamate. At 4 hr and 18 hr after exposure to either compound there was extensive depletion of the reduced folate coenzymes, which generally corresponded to the extent of inhibition of glycine and deoxyuridine incorporation. This was accompanied by an increase of the cellular dihydrofolate and 10-formyldihydrofolate. In the H35 cells the effect of methotrexate polyglutamates on the reduced folate coenzyme pools was restricted to dividing cultures, because the reduced folate coenzymes were not depleted in confluent cultures. The results demonstrate that the methotrexate and methotrexate polyglutamates that initially accumulate within dividing H35 cells readily inhibit dihydrofolate reductase but are not adequate to inhibit thymidylate synthase and prevent the depletion of reduced folate coenzymes. Thus, inhibition of de novo glycine and deoxyuridine incorporation into DNA as a result of dihydrofolate reductase inhibitors appears to be closely related to a reduction in the intracellular concentration of 10-formyltetrahydrofolate and 5,10-methylenetetrahydrofolate, the respective folate coenzymes for de novo purine and thymidylate synthesis.

Topics: Animals; Folic Acid; Folic Acid Antagonists; Leucovorin; Liver Neoplasms, Experimental; Methotrexate; Tetrahydrofolates; Tumor Cells, Cultured