5-10-methylenetetrahydrofolic-acid and 5-methyltetrahydrofolate

Trypanosomatid parasitic protozoans of the genus Leishmania are autotrophic for both folate and unconjugated pteridines. Leishmania salvage these metabolites from their mammalian hosts and insect vectors through multiple transporters. Within the parasite, folates are reduced by a bifunctional DHFR (dihydrofolate reductase)-TS (thymidylate synthase) and by a novel PTR1 (pteridine reductase 1), which reduces both folates and unconjugated pteridines. PTR1 can act as a metabolic bypass of DHFR inhibition, reducing the effectiveness of existing antifolate drugs. Leishmania possess a reduced set of folate-dependent metabolic reactions and can salvage many of the key products of folate metabolism from their hosts. For example, they lack purine synthesis, which normally requires 10-formyltetrahydrofolate, and instead rely on a network of purine salvage enzymes. Leishmania elaborate at least three pathways for the synthesis of the key metabolite 5,10-methylene-tetrahydrofolate, required for the synthesis of thymidylate, and for 10-formyltetrahydrofolate, whose presumptive function is for methionyl-tRNAMet formylation required for mitochondrial protein synthesis. Genetic studies have shown that the synthesis of methionine using 5-methyltetrahydrofolate is dispensable, as is the activity of the glycine cleavage complex, probably due to redundancy with serine hydroxymethyltransferase. Although not always essential, the loss of several folate metabolic enzymes results in attenuation or loss of virulence in animal models, and a null DHFR-TS mutant has been used to induce protective immunity. The folate metabolic pathway provides numerous opportunities for targeted chemotherapy, with strong potential for 'repurposing' of compounds developed originally for treatment of human cancers or other infectious agents.

Topics: Amino Acid Oxidoreductases; Animals; Carrier Proteins; Folic Acid; Host-Parasite Interactions; Iron-Sulfur Proteins; Leishmania; Metabolic Networks and Pathways; Methionine; Multienzyme Complexes; Pteridines; Purines; Tetrahydrofolate Dehydrogenase; Tetrahydrofolates; Thymidylate Synthase; Transferases

Calcium folinate (leucovorin), which is converted in vivo into biologically active folate, enhances the potency of 5-fluorouracil (5-FU)-based chemotherapy in colorectal cancer. A common dosage of leucovorin in adjuvant and palliative settings is 60 mg/m(2). The aim was to determine the levels of tetrahydrofolate (THF), 5,10-methylenetetrahydrofolate (methyleneTHF), and 5-methyltetrahydrofolate (methylTHF) in tumour and mucosa of colorectal cancer patients who received different dosages of leucovorin intravenously at time of surgery.. Eighty patients scheduled for colorectal resection with indication of colorectal cancer were randomised into four groups to receive leucovorin at 0, 60, 200, or 500 mg/m(2), respectively. Blood samples were taken 10 and 30 min after leucovorin administration. Biopsy samples from tumour and mucosa were collected and snap-frozen at surgery. The levels of THF, methyleneTHF, and methylTHF in tumour and mucosa were assessed by liquid chromatography electrospray ionisation tandem mass spectrometry (LC-MS/MS) and the results were related to clinical diagnosis and therapeutic regimens.. The folate levels in tissue revealed extensive inter-individual variability. The mean methyleneTHF value for the four treatment groups were 880, 1,769, 3,024 and 3,723 pmol/gww. Only half of the patients who received 60 mg/m(2) leucovorin had higher levels of methyleneTHF in tumour than patients who received 0 mg/m(2) leucovorin. Rectal cancer patients had significantly lower levels of methyleneTHF compared with colon cancer patients.. There was a large inter-patient variability of tissue folate levels in colorectal cancer patients after supplementation with leucovorin at standardised dosage. High leucovorin doses were needed to exceed baseline methyleneTHF values, especially in rectal cancer patients. The results indicate that the standardised leucovorin dose may be insufficient to attain the full antitumour effect of 5-FU. Further studies are needed to establish whether higher dosage yields a better treatment response.

Topics: Adult; Aged; Aged, 80 and over; Chromatography, Liquid; Colorectal Neoplasms; Dose-Response Relationship, Drug; Female; Humans; Leucovorin; Male; Middle Aged; Spectrometry, Mass, Electrospray Ionization; Tandem Mass Spectrometry; Tetrahydrofolates; Vitamin B Complex

To compare the efficacy of a diet rich in natural folate and of two different folic acid supplementation protocols in subjects with "moderate" hyperhomocysteinemia, also taking into account C677T polymorphism of 5,10-methylenetetrahydrofolate reductase (MTHFR) gene.. We performed a 13 week open, randomized, double blind clinical trial on 149 free living persons with mild hyperhomocyteinemia, with daily 200 μg from a natural folate-rich diet, 200 μg [6S]5-methyltetrahydrofolate (5-MTHF), 200 μg folic acid or placebo. Participants were stratified according to their MTHFR genotype.. Homocysteine (Hcy) levels were reduced after folate enriched diet, 5-MTHF or folic acid supplementation respectively by 20.1% (p < 0.002), 19.4% (p < 0.001) and 21.9% (p < 0.001), as compared to baseline levels and significantly as compared to placebo (p < 0.001, p < 0.002 and p < 0.001, respectively for enriched diet, 5-MTHF and folic acid). After this enriched diet and the folic acid supplementation, Hcy in both genotype groups decreased approximately to the same level, with higher percentage decreases observed for the TT group because of their higher pre-treatment value. Similar results were not seen by genotype for 5-MTHF. A significant increase in RBC folate concentration was observed after folic acid and natural folate-rich food supplementations, as compared to placebo.. Supplementation with natural folate-rich foods, folic acid and 5-MTHF reached a similar reduction in Hcy concentrations.

Topics: Adult; Dietary Supplements; Double-Blind Method; Female; Folic Acid; Genotype; Homocysteine; Humans; Hyperhomocysteinemia; Male; Methylenetetrahydrofolate Reductase (NADPH2); Middle Aged; Polymorphism, Genetic; Severity of Illness Index; Tetrahydrofolates; Vitamin B Complex

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

Methylenetetrahydrofolate reductase (MTHFR) catalyzes the irreversible conversion of 5,10-methylene-tetrahydrofolate (THF) to 5-methyl-THF, thereby committing one-carbon units to the methionine cycle. While MTHFR has long been known to be allosterically inhibited by S-adenosylmethionine (SAM), only relatively recently has N-terminal multisite phosphorylation been shown to provide an additional layer of regulation. In vitro, the multiply phosphorylated form of MTHFR is more sensitive to allosteric inhibition by SAM. Here we sought to investigate the kinases responsible for MTHFR multisite phosphorylation and the physiological function of MTHFR phosphorylation in cells. We identified DYRK1A/2 and GSK3A/B among the kinases that phosphorylate MTHFR. In addition, we found that MTHFR phosphorylation is maintained by adequate cellular SAM levels, which are sensed through the C-terminal SAM binding domain of MTHFR. To understand the function of MTHFR phosphorylation in cells, we generated MTHFR CRISPR knockin mutant lines that effectively abolished MTHFR phosphorylation and compared them with the parental cell lines. Whereas the parental cell lines showed increased 5-methyl-THF production in response to homocysteine treatment, the knockin cell lines had high basal levels of 5-methyl-THF and did not respond to homocysteine treatment. Overall, our results suggest that MTHFR multisite phosphorylation coordinates with SAM binding to inhibit MTHFR activity in cells.

Topics: Allosteric Regulation; HCT116 Cells; HeLa Cells; Humans; MCF-7 Cells; Methylenetetrahydrofolate Reductase (NADPH2); Phosphorylation; S-Adenosylmethionine; Tetrahydrofolates

The cofactor tetrahydrofolate (THF) is used to reduce, oxidize, and transfer one-carbon (1C) units required for the synthesis of nucleotides, glycine, and methionine. Measurement of intracellular THF species is complicated by their chemical instability, signal dilution caused by variable polyglutamation, and the potential for interconversion among these species. Here, we describe a method using negative mode liquid chromatography-mass spectrometry (LC-MS) to measure intracellular folate species from mammalian cells. Application of this method with isotope-labeled substrates revealed abiotic interconversion of THF and methylene-THF, which renders their separate quantitation particularly challenging. Chemical reduction of methylene-THF using deuterated sodium cyanoborohydride traps methylene-THF, which is unstable, as deuterated 5-methyl-THF, which is stable. Together with proper sample handling and LC-MS, this enables effective measurements of five active folate pools (THF, 5-methyl-THF, methylene-THF, methenyl-THF/10-formyl-THF, and 5-formyl-THF) representing the biologically important 1C oxidation states of THF in mammalian cells. Graphical abstract Chemical derivatization with deuterated cyanoborohydride traps unstable methylene-THF as isotope-labeled 5-methyl-THF, enabling accurate quantification by LC-MS.

Topics: Cell Culture Techniques; Chromatography, Liquid; Folic Acid Antagonists; HEK293 Cells; Humans; Leucovorin; Mass Spectrometry; Methotrexate; Tetrahydrofolates

5,10-Methylenetetrahydrofolate reductase (MTHFR) catalyzes the NADPH-dependent reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate using FAD as the cofactor. Severe MTHFR deficiency is the most common inborn error of folate metabolism, resulting in hyperhomocysteinemia and homocystinuria. Approximately 70 missense mutations have been described that cause severe MTHFR deficiency, however, in most cases their mechanism of dysfunction remains unclear. Few studies have investigated mutational specific defects; most of these assessing only activity levels from a handful of mutations using heterologous expression. Here, we report the in vitro expression of 22 severe MTHFR missense mutations and two known single nucleotide polymorphisms (p.Ala222Val, p.Thr653Met) in human fibroblasts. Significant reduction of MTHFR activity (<20 % of wild-type) was observed for five mutant proteins that also had highly reduced protein levels on Western blot analysis. The remaining mutations produced a spectrum of enzyme activity levels ranging from 22-122 % of wild-type, while the SNPs retained wild-type-like activity levels. We found increased thermolability for p.Ala222Val and seven disease-causing mutations all located in the catalytic domain, three of which also showed FAD responsiveness in vitro. By contrast, six regulatory domain mutations and two mutations clustering around the linker region showed increased thermostability compared to wild-type protein. Finally, we confirmed decreased affinity for NADPH in individual mutant enzymes, a result previously described in primary patient fibroblasts. Our expression study allows determination of significance of missense mutations in causing deleterious loss of MTHFR protein and activity, and is valuable in detection of aberrant kinetic parameters, but should not replace investigations in native material.

Topics: Amino Acid Metabolism, Inborn Errors; Catalytic Domain; Fibroblasts; Genotype; Homocystinuria; Humans; Hyperhomocysteinemia; Kinetics; Methylenetetrahydrofolate Reductase (NADPH2); Muscle Spasticity; Mutant Proteins; Mutation, Missense; NADP; Polymorphism, Single Nucleotide; Psychotic Disorders; Tetrahydrofolates

The hydroxymethyl group of serine is a primary source of tetrahydrofolate (THF)-activated one-carbon units that are required for the synthesis of purines and thymidylate and for S-adenosylmethionine (AdoMet)-dependent methylation reactions. Serine hydroxymethyltransferase (SHMT) catalyzes the reversible and THF-dependent conversion of serine to glycine and 5,10-methylene-THF. SHMT is present in eukaryotic cells as mitochondrial SHMT and cytoplasmic (cSHMT) isozymes that are encoded by distinct genes. In this study, the essentiality of cSHMT-derived THF-activated one-carbons was investigated by gene disruption in the mouse germ line. Mice lacking cSHMT are viable and fertile, demonstrating that cSHMT is not an essential source of THF-activated one-carbon units. cSHMT-deficient mice exhibit altered hepatic AdoMet levels and uracil content in DNA, validating previous in vitro studies that indicated this enzyme regulates the partitioning of methylenetetrahydrofolate between the thymidylate and homocysteine remethylation pathways. This study suggests that mitochondrial SHMT-derived one-carbon units are essential for folate-mediated one-carbon metabolism in the cytoplasm.

Topics: Animals; Cell Nucleus; Cytoplasm; Female; Fibroblasts; Gene Expression Regulation, Enzymologic; Glycine Hydroxymethyltransferase; Male; Mice; Mice, Transgenic; Mitochondria; Models, Biological; Models, Genetic; Tetrahydrofolates

Topics: Chromatography, High Pressure Liquid; Drug Monitoring; Folic Acid Antagonists; Humans; Leucovorin; Methotrexate; Neoplasms; Tetrahydrofolates

The folate coenzyme 5,10-methylenetetrahydrofolate is an important folate metabolite which cannot be determined directly by HPLC near neutral pH because it dissociates to formaldehyde and tetrahydrofolate. A method for the determination of 5,10-methylenetetrahydrofolate in liver is described. This method involves (1) determination of liver 5-methyltetrahydrofolate; (2) chemical reduction of liver 5,10-methylenetetrahydrofolate (stabilized at pH 10) to 5-methyltetrahydrofolate; and (3) determination of total liver 5-methyltetrahydrofolate. Subtraction of (1) from (3) gives the concentration of 5,10-methylenetetrahydrofolate in liver.

Topics: Animals; Chromatography, High Pressure Liquid; Liver; Male; Mice; Oxidation-Reduction; Rats; Rats, Sprague-Dawley; Tetrahydrofolates

Methylenetetrahydrofolate reductase catalyzes the reduction of N(5), N(10)-methylenetetrahydrofolate to N(5)-methyltetrahydrofolate. Because this substrate is unstable and dissociates spontaneously into formaldehyde and tetrahydrofolate, the customary method to assay the catalytic activity of this enzyme has been to measure the oxidation of [14C]N(5)-methyltetrahydrofolate to N(5), N(10)-methylenetetrahydrofolate and quantify the [14C]formaldehyde that dissociates from this product. This report describes a very sensitive radioenzymatic assay that measures directly the reductive catalysis of N(5),N(10)-methylenetetrahydrofolate. The radio-labeled substrate, [14C]N(5),N(10)-methylenetetrahydrofolate, is prepared by condensation of [C(14)]formaldehyde with tetrahydrofolate and the stability of this substrate is maintained for several months by storage at -80 degrees C in a pH 9.5 buffer. Partially purified methylenetetrahydrofolate reductase from rat liver, incubated with the radio-labeled substrate and the cofactors, NADPH and FAD at pH 7. 5, generates [14C]N(5)-methyltetrahydrofolate, which is stable and partitions into the aqueous phase after the assay is terminated with dimedone and toluene. A K(m) value of 8.2 microM was obtained under conditions of increasing substrate concentration to ensure saturation kinetics. This method is simple, very sensitive and measures directly the reduction of N(5), N(10)-methylenetetrahydrofolate to N(5)-methyltetrahydrofolate, which is the physiologic catalytic pathway for methylenetetrahydrofolate reductase.

Topics: Animals; Carbon Radioisotopes; Catalysis; Chromatography, Thin Layer; Dose-Response Relationship, Drug; Flavin-Adenine Dinucleotide; Humans; Kinetics; Liver; Methylenetetrahydrofolate Reductase (NADPH2); NADP; Oxidation-Reduction; Oxidoreductases Acting on CH-NH Group Donors; Rats; S-Adenosylhomocysteine; S-Adenosylmethionine; Sensitivity and Specificity; Tetrahydrofolates; Tumor Cells, Cultured

The identification, expression, and assay of two Saccharomyces cerevisiae genes encoding methylenetetrahydrofolate reductases (MTHFR) is described. MTHFR catalyzes the reduction of 5, 10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, used to methylate homocysteine in methionine synthesis. The MET12 gene is located on chromosome XVI and encodes a protein of 657 amino acids. The MET13 gene is located on chromosome VII and encodes a protein of 599 amino acids. The deduced amino acid sequences of these two genes are 34% identical to each other and 32-37% identical to the human MTHFR. A phenotype for the single disruption of MET12 was not observed, however, single disruption of MET13 resulted in methionine auxotrophy. Double disruption of both MET12 and MET13 also resulted in methionine auxotrophy. Growth of the methionine auxotrophs was supported by both methionine and S-adenosylmethionine. Transcripts of both MET12 and MET13 were detected in total RNA from wild type cells grown in the presence or absence of methionine. The methionine requirement of the met12 met13 double disruptant was complemented by plasmid-borne MET13, but not MET12 even when a multicopy plasmid was used. Furthermore, overexpression of the human MTHFR in the met12 met13 double disruptant complemented the methionine auxotrophy of this strain. In contrast, overexpression of the Escherichia coli metF gene did not complement the methionine requirement of met12 met13 cells. Assays for MTHFR in crude extracts and expression of the yeast proteins in Escherichia coli verified that both MET12 and MET13 encode functional MTHFR isozymes.

Topics: Amino Acid Sequence; Base Sequence; Cloning, Molecular; Enzyme Activation; Escherichia coli; Gene Deletion; Gene Expression Profiling; Genes, Fungal; Genetic Complementation Test; Humans; Isoenzymes; Methionine; Methylenetetrahydrofolate Reductase (NADPH2); Molecular Sequence Data; Oxidoreductases Acting on CH-NH Group Donors; Physical Chromosome Mapping; Recombinant Proteins; RNA, Messenger; Saccharomyces cerevisiae; Sequence Alignment; Tetrahydrofolates; Vitamin K

We examined the effect of enteric infusion of 5,10-methylenetetrahydrofolate (5,10-CH2-H4PteGlu), tetrahydrofolate (H4PteGlu), 10-formyltetrahydrofolate (10-HCO-H4PteGlu), and 5-methyltetrahydrofolate (5-CH3-H4PteGlu) on concentrations of plasma 5-CH3-H4PteGlu in rats. Concentrations of plasma 5-CH3-H4PteGlu rapidly decreased during continuous bile diversion, whereas initial levels of plasma 5-CH3-H4PteGlu were maintained by means of enteric infusion of 5-CH3-H4PteGlu at a dose of 3 nmol/h, which was approximately equal to total secretion of bile folates. Plasma 5-CH3-H4PteGlu levels were also maintained by the infusion of 5,10-CH2-H4PteGlu, H4PteGlu, or 10-HCO-H4PteGlu at the same dose as 5-CH3-H4PteGlu. The results indicate that folates secreted into the bile are reabsorbed through the intestine to regulate plasma concentrations of 5-CH3-H4PteGlu. We examined the secretion kinetics of bile folates after the intravenous injection of 5-CH3-H4PteGlu or folic acid (PteGlu) at 1 mg/kg body wt. When 5-CH3-H4PteGlu was injected, the bile secretion of folates other than 5-CH3-H4PteGlu increased by < 5 times the base level, whereas that of bile 5-CH3-H4PteGlu markedly increased by approximately 30 times. When PteGlu was given, the bile secretion of nonmethylated tetrahydrofolates markedly increased by 15-27 times, whereas that of 5-CH3-H4PteGlu increased by approximately 7 times. The results suggest that oxidized folates may be one of the sources for bile nonmethylated tetrahydrofolates. It is concluded that nonmethylated tetrahydrofolates in bile play an important role in folate homeostasis through enterohepatic circulation, together with 5-CH3-H4PteGlu.

Topics: Animals; Bile; Female; Folic Acid; Homeostasis; Injections, Intravenous; Intestines; Kinetics; Liver Circulation; Osmolar Concentration; Rats; Rats, Sprague-Dawley; Regional Blood Flow; Tetrahydrofolates

DNA photolyase from Escherichia coli contains folate ([6S]-5,10-CH(+)-H4Pte(Glu)n = 3-6) and reduced FAD. The folate chromophore acts as an antenna, harvesting light energy which is transferred to the reduced flavin where DNA repair occurs. The folate binding stereospecificity of the enzyme was investigated by reconstituting the apoenzyme with [6R,S]-5,10-CH(+)-H4folate and reduced FAD. The isomer composition of [methyl-3H]-5-CH3-H4folate, released into solution upon reduction of the reconstituted enzyme with [3H]NaBH4, was analyzed by enzymatic and chiral chromatographic methods. Both methods showed that the reconstituted enzyme contained nearly equimolar amounts of [6R]- and [6S]-5,10-CH(+)-H4folate.

Topics: Apoenzymes; Deoxyribodipyrimidine Photo-Lyase; Escherichia coli; Flavin-Adenine Dinucleotide; Folic Acid; Stereoisomerism; 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

[6RS]Leucovorin (5-formyltetrahydrofolate; 5-CHO-H4PteGlu) administered in different regimens in combination with 5-fluorouracil (FUra) has increased the response rates to FUra in patients with colon adenocarcinoma. Using preclinical models of human colon adenocarcinomas as xenografts in immune-deprived mice, the effect of the rate of administration of racemic [6RS]leucovorin on the concentration-time profile of reduced folates in plasma, size of intratumor pools of 5,10-methylenetetrahydrofolates (CH2-H4PteGlun) and tetrahydrofolates (H4PteGlun), and the distribution of their polyglutamate species have been examined. Bolus injection i.v., or 4-h or 24-h infusion of [6RS]leucovorin (500 mg/m2) yielded similar concentration profiles of the biologically active [6S] and inactive [6R] isomers of 5-CHO-H4-PteGlu and 5-methyltetrahydrofolate (5-CH3-H4PteGlu) in mouse plasma to those previously reported in humans, but with more rapid elimination half-lives (t1/2 = 11 to 16 min, 23 to 41 min, and 30 to 35 min, respectively). Thus, reduced folates remained elevated in plasma during the period of [6RS]leucovorin administration. In HxELC2 and HxGC3 tumors, pools of CH2-H4PteGlun and H4PteGlun were increased from 350% to 700% of control, but only during [6RS]leucovorin infusion. Intracellular levels subsequently declined rapidly, similar to the loss of reduced folates from plasma. Increasing the rate of [6RS]leucovorin delivery by decreasing the time for administration from a 24-h to a 4-h infusion did not further increase the intratumor pools of CH2-H4PteGlun and H4PteGlun, suggesting saturation in the cellular metabolism of [6RS]leucovorin. In HxGC3 tumors, CH2-H4PteGlu4-5 were elevated more rapidly than in line HxELC2, which accumulated predominantly a shorter chain length species following i.v. bolus injection. During the 4-h infusion schedule, di- and triglutamate species in particular accumulated in both tumors with no elevation in CH2-H4PteGlu5 until the infusion was discontinued, when this species increased as the shorter chain length forms were declining. However, during the 24-h infusion of [6RS]leucovorin, CH2-H4PteGlu3-5 were elevated in tumors. Since these species have been reported to increase the binding affinity of [6-3H]5-fluorodeoxyuridine monophosphate ([6-3H]FdUMP) to thymidylate synthase, and intratumor pools of CH2-H4PteGlun and H4PteGlun were elevated during the 24-h infusion of [6RS]leucovorin, this was considered to be the preferred schedule

Topics: Adenocarcinoma; Animals; Colonic Neoplasms; Female; Floxuridine; Half-Life; Humans; Injections, Intravenous; Leucovorin; Mice; Mice, Inbred CBA; Tetrahydrofolates; Thymidylate Synthase; Time Factors

The quantitation of carbon flow through the folate-dependent one-carbon pool in regard to the synthesis of methionine from the amino acid precursor, serine, was determined in rat liver. Utilizing duodenal cannulated rats and in vivo tracer kinetic techniques where [3-14C]serine was continuously infused at a rate of 3.32 microCi/h, a steady-state (plateau) specific radioactivity was achieved within 200 min from the onset of the infusion period. This resulted in an irreversible loss rate of 431 +/- 12 mumol/h for hepatic serine. In conjunction with the specific radioactivity measurements of hepatic methionine, the percentage of the total entry into the hepatic methionine methyl carbon pool that came from serine (i.e., transfer quotient) was calculated to be 51.7 +/- 5.2%. Similar experiments utilizing [methyl-3H]methionine as the infusate resulted in a value of 112 +/- 6 mumol/h for the irreversible loss rate of hepatic methionine. Using the irreversible loss rate of methionine and the transfer quotient to methionine from serine, the flux of the beta-carbon of serine to remethylate homocysteine and generate methionine was calculated to be 57.9 mumol/h. These results not only present a methodology for the determination of folate-dependent carbon flow in vivo, but also demonstrate the high degree to which the homocysteine moiety of methionine is conserved in vivo to meet the methylation requirements in the rat.

Topics: Animals; Biological Transport; Carbon; Homocysteine; Liver; Male; Methionine; Rats; Rats, Inbred Strains; Serine; Tetrahydrofolates; Time Factors

Four hr infusions i.v. of [6RS]5-formyltetrahydrofolate ([6RS]5-CHO-H4PteGlu; 500 mg/m2) and [6RS]5-methyltetrahydrofolate ([6RS]5-CH3-H4PteGlu; 500 mg/m2) were compared for their relative effects on expansion of pools of 5,10-methylenetetrahydrofolates (CH2-H4PteGlun) and tetrahydrofolates (H4PteGlun) in two human colon adenocarcinoma xenografts in mice. Expansion of these pools by 253-661% of control and increase in predominance of di-, tri-, and tetra-glutamate species were observed during [6RS]5-CHO-H4PteGlu infusion. In contrast, only modest pool size expansion (148-164% of control) and limited modulation of polyglutamate species were detected in four tumor lines during infusion with [6RS]5-CH3-H4PteGlu. The data suggest that [6RS]5-CH3-H4PteGlu is less effective than [6RS]5-CHO-H4PteGlu as a precursor for pools of CH2-H4PteGlun and H4PteGlun in colon tumors.

Topics: Adenocarcinoma; Animals; Biotransformation; Cell Line; Colonic Neoplasms; Female; Formyltetrahydrofolates; Humans; Kinetics; Mice; Mice, Inbred CBA; Neoplasm Transplantation; Pteroylpolyglutamic Acids; Tetrahydrofolates; Transplantation, Heterologous

Topics: 5,10-Methylenetetrahydrofolate Reductase (FADH2); Alcohol Oxidoreductases; Animals; Chemical Phenomena; Chemistry; Deuterium; Liver; Oxidation-Reduction; Protons; Solvents; Swine; Tetrahydrofolates