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PubMed1972

Regulation of C metabolism by L-methionine in Saccharomyces cerevisiae.

1. The concentrations of folate derivatives in aerobic cultures of Saccharomyces cerevisiae (A.T.C.C. 9763) were determined by microbiological assay employing Lactobacillus casei (A.T.C.C. 7469) and Pediococcus cerevisiae (A.T.C.C. 8081). Cells cultured in media lacking l-methionine contained higher concentrations of folate derivatives than cells grown in the same media supplemented with 2.5mumol of l-methionine/ml. The concentrations of highly conjugated derivatives were also decreased by supplementing the growth medium with l-methionine. 2. DEAE-cellulose column chromatography of extracts prepared from cells grown under these conditions revealed that the concentrations of methylated tetrahydrofolates were drastically decreased by the methionine supplement. Smaller decreases were also observed in the concentrations of formylated and unsubstituted derivatives. 3. The concentrations of four enzymes of C(1) metabolism were compared after 6h of growth in the presence and in the absence of l-methionine (2.5mumol/ml). The specific activities of formyltetrahydrofolate synthetase, methylenetetrahydrofolate reductase and serine hydroxymethyltransferase were not altered by this treatment but that of 5-methyltetrahydrofolate-homocysteine methyltransferase was decreased by approx. 65% when l-methionine was supplied. The activities of 5-methyltetrahydrofolate-homocysteine methyltransferase, serine hydroxymethyltransferase and formyltetrahydrofolate synthetase were not appreciably altered by l-methionine in vitro. In contrast this amino acid was found to inhibit the activity of methylenetetrahydrofolate reductase. 4. Feeding experiments employing sodium [(14)C]formate indicated that cells grown in the presence of exogenous methionine, although having less ability to convert formate into methionine, readily incorporated (14)C into serine and the adenosyl moiety of S-adenosylmethionine. 5. It is suggested that exogenous l-methionine controls C(1) metabolism in Saccharomyces principally by regulation of methyl-group biogenesis within the folate pool.

Alcohol Oxidoreductases↗

PubMed1975

New approach to antifolate treatment of certain cancers as demonstrated in tissue culture.

The selective toxicity of antifolates for a variety of cancers can be improved, as illustrated by the combined administration of N5-methyltetrahydrofolate and methotrexate in tissue culture. When a variety of neoplastic cell types characterized by a deficiency of vitamin B12-dependent N5-methyltetrahydrofolate methyltransferase (5-methyltetrahydropteroyl-L-glutamate:L-homocysteine S-methyltransferase, EC 2.1.1.13) and normal adult cells are grown in media containing methotrexate and either N5-methyltetrahydrofolate or N5-formyltetrahydrofolate, not only is the selective toxicity of methotrexate demonstrated, but the advantage of using N5-methyltetrahydrofolate in place of N5-formyltetrahydrofolate is also revealed. The implications and applications of this particular combination in the treatment of human cancer are discussed.

Animals↗

PubMed2003

Methenyltetrahydrofolate synthetase regulates folate turnover and accumulation.

Cellular folate deficiency impairs one-carbon metabolism, resulting in decreased fidelity of DNA synthesis and inhibition of numerous S-adenosylmethionine-dependent methylation reactions including protein and DNA methylation. Cellular folate concentrations are influenced by folate availability, cellular folate transport efficiency, folate polyglutamylation, and folate turnover specifically through degradation. Folate cofactors are highly susceptible to oxidative degradation in vitro with the exception of 5-formyltetrahydrofolate, which may be a storage form of folate. In this study, we determined the effects of depleting cytoplasmic 5-formyltetrahydrofolate on cellular folate concentrations and folate turnover rates in cell cultures by expressing the human methenyltetrahydrofolate synthetase cDNA in human MCF-7 cells and SH-SY5Y neuroblastoma. Cells with increased methenyltetrahydrofolate synthetase activity exhibited: 1) increased rates of folate turnover, 2) elevated generation of p-aminobenzoylglutamate in culture medium, 3) depressed cellular folate concentrations independent of medium folic acid concentrations, and 4) increased average polyglutamate chain lengths of folate cofactors. These data indicate that folate catabolism and folate polyglutamylation are competitive reactions that influence cellular folate concentrations, and that increased methenyltetrahydrofolate synthetase activity accelerates folate turnover rates, depletes cellular folate concentrations, and may account in part for tissue-specific differences in folate accumulation.

Biological Transport↗

PubMed2005

Structure and function of both domains of ArnA, a dual function decarboxylase and a formyltransferase, involved in 4-amino-4-deoxy-L-arabinose biosynthesis.

Modification of the lipid A moiety of lipopolysaccharide by the addition of the sugar 4-amino-4-deoxy-L-arabinose (L-Ara4N) is a strategy adopted by pathogenic Gram-negative bacteria to evade cationic antimicrobial peptides produced by the innate immune system. L-Ara4N biosynthesis is therefore a potential anti-infective target, because inhibiting its synthesis would render certain pathogens more sensitive to the immune system. The bifunctional enzyme ArnA, which is required for L-Ara4N biosynthesis, catalyzes the NAD(+)-dependent oxidative decarboxylation of UDP-glucuronic acid to generate a UDP-4'-keto-pentose sugar and also catalyzes transfer of a formyl group from N-10-formyltetrahydrofolate to the 4'-amine of UDP-L-Ara4N. We now report the crystal structure of the N-terminal formyltransferase domain in a complex with uridine monophosphate and N-5-formyltetrahydrofolate. Using this structure, we identify the active site of formyltransfer in ArnA, including the key catalytic residues Asn(102), His(104), and Asp(140). Additionally, we have shown that residues Ser(433) and Glu(434) of the decarboxylase domain are required for the oxidative decarboxylation of UDP-GlcUA. An E434Q mutant is inactive, suggesting that chemical rather than steric properties of this residue are crucial in the decarboxylation reaction. Our data suggest that the decarboxylase domain catalyzes both hydride abstraction (oxidation) from the C-4' position and the subsequent decarboxylation.

Amino Acid Sequence↗

PubMed2005

Higher plant plastids and cyanobacteria have folate carriers related to those of trypanosomatids.

Cyanobacterial and plant genomes encode proteins with some similarity to the folate and biopterin transporters of the trypanosomatid parasite Leishmania. The Synechocystis slr0642 gene product and its closest Arabidopsis homolog, the At2g32040 gene product, are representative examples. Both have 12 probable transmembrane domains, and the At2g32040 protein has a predicted chloroplast transit peptide. When expressed in Escherichia coli pabA pabB or folE, mutants, which are unable to produce or take up folates, the slr0642 protein and a modified At2g32040 protein (truncated and fused to the N terminus of slr0642) enabled growth on 5-formyltetrahydrofolate or folic acid but not on 5-formyltetrahydrofolate triglutamate, demonstrating that both proteins mediate folate monoglutamate transport. Both proteins also mediate transport of the antifolate analogs methotrexate and aminopterin, as evidenced by their ability to greatly increase the sensitivity of E. coli to these inhibitors. The full-length At2g32040 polypeptide was translocated into isolated pea chloroplasts and, when fused to green fluorescent protein, directed the passenger protein to the envelope of Arabidopsis chloroplasts in transient expression experiments. At2g32040 transcripts were present at similar levels in roots and aerial organs, indicating that the protein occurs in non-green plastids as well as chloroplasts. Insertional inactivation of At2g32040 significantly raised the total folate content of chloroplasts and lowered the proportion of 5-methyltetrahydrofolate but did not discernibly affect growth. These findings establish conservation of function among folate and biopterin transporter family proteins from three kingdoms of life.

Aminopterin↗

PubMed1975

Experimental production of elevated serum folate in dogs with intestinal blind loops. II. Nature of bacterially produced folate coenzymes in blind loop fluid.

Jejunal and ileal blind loops were constructed in separate groups of dogs and the folate coenzymes present in these loops were investigated. Although only those dogs with high blind loops had appreciably elevated folate levels active for Lactobacillus casei but not Streptococcus faecalis, every blind loop appeared to contain four folate compounds--5-formyltetrahydrofolic acid, 5-methyltetrahydrofolic acid, tetrahydrofolic acid and a compound tentatively identified as 5-formyltetrahydrofolic triglutamate. Why increased levels of 5-methyltetrahydrofolate alone are present in the serum of dogs with a high blind loop is discussed.

Animals↗

PubMed1989

Characterization of de novo folate synthesis in Pneumocystis carinii and Toxoplasma gondii: potential for screening therapeutic agents.

Drug therapy studies imply that Pneumocystis carinii and Toxoplasma gondii possess the enzymes necessary for de novo folate synthesis. To verify this, incorporation of [3H]paraaminobenzoic acid [( 3H]PABA) into reduced folates by P. carinii and T. gondii was investigated. Both organisms synthesized tritiated reduced folates. In P. carinii, 10-formyltetrahydrofolate and tetrahydrofolate, and in T. gondii, 5-formyltetrahydrofolate were the major synthesized folates. P. carinii remained metabolically active in vitro for only a few days. Because current systems for screening antipneumocystis agents are cumbersome, the utility of this assay system for screening therapeutic agents was investigated. Sulfonamides and pentamidine efficiently inhibited de novo folate synthesis in P. carinii. Inhibitors of dihydrofolate reductase such as trimethoprim and trimetrexate were poor inhibitors for P. carinii but efficient inhibitors for T. gondii. This study demonstrates the first unambiguous evidence of metabolic activity in P. carinii, and provides a potential assay for efficiently screening antipneumocystis drugs in vitro.

4-Aminobenzoic Acid↗

PubMed2003

Neither methionine nor nitrous oxide inactivation of methionine synthase affect the concentration of 5,10-methylenetetrahydrofolate in rat liver.

5,10-Methylenetetrahydrofolate occupies a key position in folate-dependent one-carbon metabolism. It is involved directly in the biosynthesis of deoxythymidine, it can be converted to 10-formyltetrahydrofolate for purine synthesis and it may be reduced to 5-methyltetrahydrofolate for methylation of homocysteine to methionine. We have developed a HPLC method for measuring 5,10-methylenetetrahydrofolate in liver and we have used this method to investigate two conditions that perturb one-carbon metabolism: 1) administration of methionine and 2) administration of the anesthetic gas, nitrous oxide (N(2)O). Rats were given 1.3 mmol/kg of methionine, and folate coenzymes in liver were measured. As expected, giving methionine resulted in an apparent increase in the concentration of 10-formyl- and tetrahydrofolate and an apparent decrease in 5-methyltetrahydrofolate concentration at 30 and 60 min. After 120 min, the concentrations of these coenzymes appeared to revert to control values. There was no apparent change in the concentration of 5,10-methylenetetrahydrofolate. Exposing rats to an atmosphere containing N(2)O results in inactivation of methionine synthase and accumulation of 5-methyltetrahydrofolate at the expense of other folate coenzymes. In liver from rats breathing N(2)O, 5-methyltetrahydrofolate increased, whereas there was no change in 5- or 10-formyltetrahydrofolates (P > 0.7 and P > 0.8, respectively). Tetrahydrofolate was not detected in liver from the N(2)O group, whereas it constituted 24% of folates in the control group. The concentration of 5,10-methylenetetrahydrofolate was not significantly affected by N(2)O (P > 0.18). These results suggest that the concentration of 5,10-methylenetetrahydrofolate is tightly regulated in liver.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗

PubMed2005

Methylenetetrahydrofolate reductase 677C->T polymorphism and folate status affect one-carbon incorporation into human DNA deoxynucleosides.

The methylenetetrahydrofolate reductase (MTHFR) 677C-->T polymorphism is thought to influence the partitioning of 1-carbon units between methylation and other components of 1-carbon metabolism and to influence the risk and etiology of several major cancers and cardiovascular disease. Our objective was to determine the effect of the MTHFR 677C-->T polymorphism and folate status on the relative rate and extent of in vivo synthesis of DNA precursors. Adequately nourished, healthy women (9 CC, 9 TT) were infused with [3-(13)C]serine and [(13)C(5)]methionine for 9 h before and after 7 wk of consumption of a low-folate diet. Blood was drawn over 5 d for monocyte DNA isolation. Isotopic enrichment of the nucleosides in DNA digests was determined by LC-MS/MS. Maximum thymidine enrichment tended to be higher (P = 0.07) in TT than in CC subjects, suggestive of marginally higher mean thymidylate synthesis. However, the subset of TT subjects who exhibited formyltetrahydrofolate in erythrocytes (an indicator of 1-carbon partitioning) had greater (P = 0.036) thymidine enrichment than CC subjects, who had no erythrocyte formyltetrahydrofolate. Purine enrichment was not affected by genotype or folate depletion. However, the deoxyadenosine to deoxyguanosine enrichment ratio was significantly higher in TT subjects, suggesting a greater relative rate of adenine synthesis. The approximately 40% greater (P = 0.012) labeling of the methyl group of methyldeoxycytidine during folate depletion suggests a change in the origin of this 1-carbon unit. This is the first time that 1-carbon incorporation into human DNA has been measured in vivo after infusion of (13)C-labeled 1-carbon precursors. These findings support the feasibility of further assessment of factors affecting deoxynucleotide synthesis and DNA methylation in human 1-carbon metabolism.

Adult↗

PubMed1991

Pharmacokinetics of leucovorin metabolites in human plasma as a function of dose administered orally and intravenously.

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.

Administration, Oral↗

PubMed1991

Design and testing of a functional group-specific DNA probe for the study of natural populations of acetogenic bacteria.

The acetogens, although phylogenetically diverse, can be characterized by their possession of the acetyl coenzyme A (acetyl-CoA) pathway for autotrophic CO2 fixation. The gene encoding formyltetrahydrofolate synthetase, a key enzyme of the acetyl-CoA pathway, was previously cloned from the thermophilic acetogen Clostridium thermoaceticum and has now been tested as a group-specific probe for acetogens. Stable hybrids were formed between the probe and single DNA fragments from eight known acetogens representing six genera. A hybrid was also formed between the probe and a DNA fragment from one sulfate reducer known to be capable of both autotrophic CO2 fixation and acetate catabolism. No such hybrid was formed between the probe and DNA from a homoacetate fermenter not known to use the acetyl-CoA pathway, with two known formyltetrahydrofolate synthetase-producing purine fermenters, or with DNA from 27 other species representing 16 genera of organisms that do not use the acetyl-CoA pathway. DNA purified from cells extracted from horse manure was also screened with the acetogen probe. Six hybrids, indicating at least six detectable acetogen "strains," were observed.

Acetyl Coenzyme A↗

PubMed2002

Mitochondrial NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase is essential for embryonic development.

Folate-dependent enzymes are compartmentalized between the cytoplasm and mitochondria of eukaryotes. The role of mitochondrial folate-dependent metabolism and the extent of its contribution to cytoplasmic processes are areas of active investigation. NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase (NMDMC) catalyzes the interconversion of 5,10-methylenetetrahydrofolate and 10-formyltetrahydrofolate in mitochondria of mammalian cells, but its metabolic role is not yet clear. Its expression in embryonic tissues but not in most adult tissues as well as its stringent transcriptional regulation led us to postulate that it may play a role in embryonic development. To investigate the metabolic role of NMDMC, we used a knockout approach to delete the nmdmc gene in mice. Heterozygous mice appear healthy, but homozygous NMDMC knockout mice die in utero. At embryonic day 12.5 (E12.5), homozygous null embryos exhibit no obvious developmental defects but are smaller and pale and die soon thereafter. Mutant fetal livers contain fewer nucleated cells and lack the characteristic redness of wild-type or heterozygous livers. The frequencies of CFU-erythroid (CFU-E) and burst-forming unit-erythroid (BFU-E) from fetal livers of E12.5 null mutants were not reduced compared with those of wild-type or heterozygous embryos. It has been assumed that initiation of protein synthesis in mitochondria requires a formylated methionyl-tRNA(fmet). One role postulated for NMDMC is to provide 10-formyltetrahydrofolate as a formyl group donor for the synthesis of this formylmethionyl-tRNA(fmet). To determine if the loss of NMDMC impairs protein synthesis and thus could be a cause of embryonic lethality, mitochondrial translation products were examined in cells in culture. Mitochondrial protein synthesis was unaffected in NMDMC-null mutant cell lines compared with the wild type. These results show that NMDMC is not required to support initiation of protein synthesis in mitochondria in isolated cells but instead demonstrate an essential role for mitochondrial folate metabolism during embryonic development.

Aminohydrolases↗

PubMed1986

Biosynthesis of polyglutamates of folates.

Folylpolyglutamate synthetase (FPGS) catalyzes the synthesis of the poly-gamma-glutamate forms of tetrahydrofolate and its co-enzyme adducts, as well as of the folate-analogue drugs. This paper reviews current knowledge of the preparations of FPGS from mammalian sources (rat, hog, mouse, and beef liver). Kinetic constants for the substrates and activators of FPGS are compared. Tetrahydrofolate and 5-formyltetrahydrofolate are excellent substrates for the enzyme. The Km values for the antifolates and their 7-hydroxy metabolites are much higher than those for the tetrahydrofolates. Aminopterin has higher activity with FPGS than does methotrexate, which partially explains its greater toxicity. 5-Formyltetrahydrofolate, which is used as a rescue agent in high-dose methotrexate-rescue chemotherapy, is a better alternate substrate of FPGS than is methotrexate and therefore is a potent competitive inhibitor of the glutamylation of methotrexate. Thus, low concentrations of the rescue agent prevent formation of cytotoxic polyglutamates of methotrexate. The pathway of the reaction is the addition of a glutamate residue to the terminal gamma-carboxyl of the pteridine substrate. That longer folylpolyglutamates are poorer substrates possibly is a result of this addition pathway. Pteroic acid activates FPGS by lowering the Km value of the pteridine substrate. It also greatly increases the activity of the synthetase at physiological pH values.

Animals↗

PubMed2004

Characterization of a folate transporter in HeLa cells with a low pH optimum and high affinity for pemetrexed distinct from the reduced folate carrier.

Studies were undertaken to characterize a low pH transport activity in a reduced folate carrier (RFC)-null HeLa-derived cell line (R5). This transport activity has a 20-fold higher affinity for pemetrexed (PMX; Kt, approximately 45 nmol/L) than methotrexate (MTX; Kt, approximately 1 micromol/L) with comparable Vmax values. The Ki values for folic acid, ZD9331, and ZD1694 were approximately 400-600 nmol/L, and the Ki values for PT523, PT632, and trimetrexate were >50 micromol/L. The transporter is stereospecific and has a 7-fold higher affinity for the 6S isomer than the 6R isomer of 5-formyltetrahydrofolate but a 4-fold higher affinity for the 6R isomer than the 6S isomer of dideazatetrahydrofolic acid. Properties of RFC-independent transport were compared with transport mediated by RFC at low pH using HepG2 cells, with minimal constitutive low pH transport activity, transfected to high levels of RFC. MTX influx Kt was comparable at pH 7.4 and pH 5.5 (1.7 versus 3.8 micromol/L), but Vmax was decreased 4.5-fold. There was no difference in the Kt for PMX (approximately 1.2 micromol/L) or the Ki for folic acid (approximately 130 micromol/L) or PT523 ( approximately 0.2 micromol/L) at pH 7.4 and pH 5.5. MTX influx in R5 and HepG2 transfectants at pH 5.5 was trans-stimulated in cells loaded with 5-formyltetrahydrofolate, inhibited by Cl- (HepG2-B > R5), Na+ independent, and uninhibited by energy depletion. Hence, RFC-independent low pH transport activity in HeLa R5 cells is consistent with a carrier-mediated process with high affinity for PMX. Potential alterations in protonation of RFC or the folate molecule as a function of pH do not result in changes in affinity constants for antifolates. Whereas both activities at low pH have similarities, they can be distinguished by folic acid and PT523, agents for which they have very different structural specificities.

Antimetabolites, Antineoplastic↗

PubMed2006

The inverse relationship between reduced folate carrier function and pemetrexed activity in a human colon cancer cell line.

Pemetrexed, a new generation antifolate recently approved for the treatment of mesothelioma and non-small cell lung cancer, is an excellent substrate for the reduced folate carrier (RFC). To explore the carrier's effect on pemetrexed activity, RFC was inactivated in HCT-15 colon cancer cells by mutagenesis and PT632 selective pressure. A clone (PT1) was obtained with a glycine to arginine substitution at amino acid 401, resulting in the loss of RFC function. PT1 cells were resistant to PT632 (178-fold), methotrexate (4-fold), and ZD1694 (Tomudex, raltitrexed; 20-fold), but were 3-fold collaterally sensitive to pemetrexed when grown in 25 nmol/L of 5-formyltetrahydrofolate. PT1 cells transfected with wild-type RFC had antifolate sensitivities comparable to that of wild-type HCT-15 cells, indicating that the RFC mutation was the sole basis for resistance. Folate pools were contracted in PT1 cells by 32% or 60%, as measured by radiolabeling intracellular folates or by an enzyme binding assay, respectively. This was reflected in marked (6.5-fold) collateral sensitivity to trimetrexate. The initial uptake of pemetrexed in PT1 cells was markedly reduced ( approximately 85%) but intracellular pemetrexed levels increased to approximately 60% and approximately 70% to that of wild-type cells after 2 hours and 6 days, respectively. There was increased pemetrexed inhibition of glycinamide ribonucleotide transformylase and, to a lesser extent, thymidylate synthase in PT1 cells growing in 5-formyltetrahydrofolate based on nucleoside protection analyses. Hence, loss of RFC function leads to collateral sensitivity to pemetrexed in HCT-15 cells, likely due to cellular folate pool contraction resulting in partial preservation of pemetrexed polyglutamylation and increased target enzyme inhibition. micro

Amino Acid Substitution↗

PubMed1972

The mechanism of folate transport in rabbit reticulocytes.

Folate transport in phenylhydrazine-induced rabbit reticulocytes was studied with the non-metabolized folate-analog, methotrexate. The time-course of methotrexate uptake into a mixed population of reticulocytes and mature erythrocytes is a two-component process consisting of a small, but rapid, initial uptake phase followed by a much slower uptake component which remains essentially constant over the period of observation. The velocity of the latter uptake component is directly proportional to the per cent reticulocytes and appears to represent a unidirectional influx of methotrexate into these cells. Uptake of methotrexate into reticulocytes was found to have the following characteristics: (a) temperature sensitivity, Q(10) of 4; (b) uptake velocity as a function of the extracellular methotrexate concentration approximated Michaelis-Menten kinetics with a maximum transport velocity of 48 pmoles/min per g dry wt; the extracellular methotrexate level at which the uptake velocity was one-half maximum was 1.4 muM; (c) 5-formyltetrahydrofolate markedly inhibited methotrexate uptake but pteroylglutamic acid inhibition was weak; (d) uptake was stimulated in cells preincubated with 5-formyltetrahydrofolate, indicative of hetero-exchange diffusion; (e) uptake was independent of extracellular sodium but was inhibited by anions including nitrate, phosphate, and glucose-6-phosphate; (f) uptake was enhanced by azide plus iodoacetate. These data indicate that folate transport in rabbit reticulocytes is mediated by a carrier mechanism which disappears with reticulocyte maturation. The mechanism of folate transport in rabbit reticulocytes is qualitatively similar to tumor cells previously studied; both appear to have an energy-dependent mechanism limiting folate uptake, and influx in both is inhibited by structurally unrelated inorganic and organic anions. These studies suggest that circulating pteroylglutamic acid is of little importance in meeting the folate requirements of folate-dependent tissues and raise the possibility that clinical conditions associated with alterations in the anionic composition of the blood may be accompanied by impaired utilization of the folates.

Animals↗

PubMed1988

Plasma and tumor tissue pharmacology of high-dose intravenous leucovorin calcium in combination with fluorouracil in patients with advanced colorectal carcinoma.

Plasma pharmacokinetics of high-dose (500 mg/m2) leucovorin calcium (dl-5-formyltetrahydrofolic acid [dl-CF]) and fluorouracil (FUra) have been evaluated in patients with advanced colorectal cancer treated with the combination of FUra and dl-CF by two different intravenous (IV) schedules: (A) In patients with no prior chemotherapy, dl-CF was administered by a two-hour IV infusion and FUra by rapid IV injection one hour after the start of the dl-CF infusion and (B) in previously treated patients, dl-CF and FUra were administered by five-day continuous IV infusion (CI). Following the two-hour infusion of dl-CF, mean peak plasma concentration and elimination half-life of I-5-formyltetrahydrofolic acid (I-CF) were 24 +/- 6 mumol/L and 0.8 +/- 0.1 hour, respectively. CI of dl-CF over five days yielded a mean steady-state plasma level of I-CF of only 1.2 +/- 0.5 mumol/L. Peak and steady-state plasma concentrations of the metabolite 5-methyl tetrahydrofolic acid were comparable in the two schedules (17 +/- 8 mumol/L for the two-hour infusion and 12 +/- 5 mumol/L for the CI). Areas under the concentration v time curve (AUC) of total reduced folates were significantly greater under conditions of CI: 89.0 v 16.7 mmol/L/min for the two-hour infusion. In tumor tissue, 5,10-methylenetetrahydrofolate increased eight-fold two to four hours following the two-hour infusion and two-fold during the CI of dl-CF and FUra. Inhibition of thymidylate synthase (dTMP-S) by the two-hour and CI infusion schedules were 66% v 39%, respectively. The observed differences in the intracellular dTMP-S folate cofactor pools and the degree of inhibition of dTMP-S achieved in patients treated by two different schedules may be due to differences in the biochemical properties and/or to differences in the modulation of FUra metabolism by folate of tumor tissues obtained from newly diagnosed and previously treated patients.

Antineoplastic Combined Chemotherapy Protocols↗

PubMedDate not supplied

A validated liquid chromatographic method for determining folates in vegetables, milk powder, liver, and flour.

A liquid chromatographic (LC) method was elaborated for determining folates in foods. Folates were extracted by homogenizing in buffer and heat treatment. A portion was incubated with an enzyme preparation containing conjugase, amylase, and protease. After purification by affinity chromatography, folate monoglutamates were determined by reversed-phase LC with fluorescence and diode array detection. Gradient elution with phosphate buffer and acetonitrile was used to separate vitamers. The most abundant folate forms naturally present in foods were detected, including tetrahydrofolic acid, 5-methyltetrahydrofolic acid, and 5-formyltetrahydrofolic acid. 10-Formylfolic acid could be detected by applying a second fluorescence detector. Folic acid, used for fortification, might also be quantitated with this system. The difference between folate concentrations in sample extracts, with and without treatment of conjugase, is a measure of the quantity of polyglutamates in the food matrixes. An additional treatment with conjugase, amylase, and protease reflects the amount of matrix-bound folates. The LC system gave a linear response over the range 0-100 ng/mL. Detection limit for these compounds were 7 pg/mL for tetrahydrofolic acid and 5-methyltetrahydrofolic acid and 59 pg/mL for 10-formylfolic acid (signal-to-noise ratio > or = 3) when 100 microL was injected. Detection limits for 5-formyltetrahydrofolic acid and folic acid were 1 ng/mL. Repeatability relative standard deviation values for separate folates in 3 candidate Certified Reference Materials (CRMs)--mixed vegetables (CRM 485), pig liver (CRM 487), and whole-meal flour (CRM 121)--and a Certified Reference Material milk powder (CRM 421) varied from 3.3 to 21.0% for the concentration range 1.8-1440 micrograms/100 g. Recoveries ranged from 73 to 109%. Use of amylase and protease was advantageous. Use of a commercially available folate-binding protein for cleanup saved time and money and was effective. Results for 5-methyltetrahydrofolic acid were in good agreement with results obtained with other LC methods. Results for total folates were lower than results obtained with microbiological methods.

Animals↗