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PubMed1981

Vitamin B12-dependent replication of L1210 mouse leukemia cells. A model system for cobalamin-folate inter-relationships.

L1210 mouse leukemia cells were made cobalamin-deficient by propagation in a medium from which cyanocobalamin was omitted and fetal bovine serum (containing protein-bound cobalamins) was replaced by bovine serum albumin. These cobalamin-deficient cells exhibited a normal replication time of 12 h, provided that the medium contained excess folate or 5-formyltetrahydrofolate. The cells responded poorly, however, to 5-methyltetrahydrofolate unless exogenous cobalamin was added. A cobalamin dependency was also observed when low levels of folate or 5-formyltetrahydrofolate were used. With 5-methyltetrahydrofolate, optimal stimulation of growth was observed with free and transcobalamin-II-bound cobalamin at 4,000 pM and 2 pM, respectively. Under cobalamin-replete conditions, cells contained 2,000 to 4,000 molecules of cobalamin/cell, and in the deficient state, this value declined to less than 10 molecules/cell; optimal replication on 5-methyltetrahydrofolate required approximately 180 molecules/cell. Cobalamin-deficient cells cultured in the absence of folate reached an arrested state from which limited replication could be induced by the addition of aquacobalamin; normal replication was induced by aquacobalamin plus 5-methyltetrahydrofolate. Results of this investigation are interpreted in terms of the requirement for tetrahydrofolate in cell replication and the production of this compound from folate and 5-formyltetrahydrofolate (via cobalamin-independent pathways) and from 5-methyltetrahydrofolate (via the cobalamin-dependent methionine synthetase).

Animals↗

PubMed1993

Biochemical and cellular pharmacology of 1843U89, a novel benzoquinazoline inhibitor of thymidylate synthase.

Studies on a series of benzoquinazoline folate analogues as inhibitors of human thymidylate synthase led to the selection of 1843U89 for further evaluation. This compound had a Ki of 90 pM versus human thymidylate synthase and was noncompetitive with (6R,S)-5,10-methylenetetrahydrofolate. It was a good substrate for the addition of the second glutamate by hog liver folylpolyglutamate synthetase, having a Vmax/Km value 7.8-fold higher than (6R,S)-tetrahydrofolate. The data indicate that 1843U89 was transported into cells via the reduced folate carrier. The Kt for 1843U89 in MOLT-4 cells was 0.33 microM, which was 3-fold lower than that for methotrexate and 16-fold lower than that for (6S-5-formyltetrahydrofolate. V/K values were 20.3 for 1843U89 versus 1.2 and 1.9 for methotrexate and (6S)-5-formyltetrahydrofolate, respectively. It was a potent inhibitor of the growth of human cells, having 50% inhibitory concentrations below 1 nM for all cell lines tested. Growth inhibition was reversed by thymidine alone, indicating that thymidylate synthase was the only site of action of this compound. Growth inhibition was not affected by (6R,S-5-formyltetrahydrofolate at concentrations below 5 microM. However, the 50% inhibitory concentration increased when the concentration in the medium was increased to 100 microM, presumably due to competition for transport. Relative to the human cell lines used, murine cell lines were 80-1300-fold less sensitive to 1843U89 and the other benzoquinazolines tested. This decreased sensitivity appeared to be due, at least in part, to decreased transport or accumulation in murine cells. Ki values for inhibition of methotrexate transport for the benzoquinazolines were 5-17-fold higher in L1210 cells than in MOLT-4 cells. 1843U89, the benzoquinazoline which was transported most efficiently and which was the most potent inhibitor of the growth of human cells, exhibited the largest difference between binding to the MOLT-4 human and L1210 murine transporter. The V/K for L1210 transport was 80-fold less than that for MOLT-4. Initial antitumor studies, using the human thymidine kinase-deficient line GC3TK- to circumvent problems associated with murine transport as well as the high circulating thymidine levels in mice, indicated that 1843U89 had marked in vivo antitumor activity.

Animals↗

PubMed2001

Methanol toxicity and formate oxidation in NEUT2 mice.

NEUT2 mice are deficient in cytosolic 10-formyltetrahydrofolate dehydrogenase (FDH; EC 1.5.1.6) which catalyzes the oxidation of excess folate-linked one-carbon units in the form of 10-formyltetrahydrofolate to CO(2) and tetrahydrofolate (Champion et al., Proc. Natl. Acad. Sci. USA 91, 11338-11342, 1994). The absence of FDH should impair the oxidation of formate via the folate-dependent pathway and as a consequence render homozygous NEUT2 mice more susceptible to methanol toxicity. Normal (CB6-F1) and NEUT2 heterozygous and homozygous mice had essentially identical LD(50) values for methanol, 6.08, 6.00, and 6.03 g/kg, respectively. Normal mice oxidized low doses of [(14)C]sodium formate (ip 5 mg/kg) to (14)CO(2) at approximately twice the rate of homozygous NEUT2 mice, indicating the presence of another formate-oxidizing system in addition to FDH. Treatment of mice with the catalase inhibitor, 3-aminotriazole (1 g/kg ip) had no effect on the rate of formate oxidation, indicating that at low concentrations formate was not oxidized peroxidatively by catalase. High doses of [(14)C]sodium formate (ip 100 mg/kg) were oxidized to (14)CO(2) at identical rates in normal and NEUT2 homozygous mice. Pretreatment with 3-aminotriazole (1 g/kg ip) in this instance resulted in a 40 and 50% decrease in formate oxidation to CO(2) in both normal and homozygous NEUT2 mice, respectively. These results indicate that mice are able to oxidize formate to CO(2) by at least three different routes: (1) folate-dependent via FDH at low levels of formate; (2) peroxidation by catalase at high levels of formate; and (3) by an unknown route(s) which appears to function at both low and high levels of formate. The implications of these observations are discussed in terms of the current hypotheses concerning methanol and formate toxicity in rodents and primates.

Animals↗

PubMed1979

Alterations of the carrier-mediated transport of an anionic solute, methotrexate, by charged liposomes in Ehrlich ascites tumor cells.

Interaction of positively charged liposomes with Ehrlich ascites tumor cells increases the bidirectional transmembrane fluxes of the anionic folic acid analog, methotrexate. Negative liposomes reduce methotrexate influx. Stimulation of methotrexate influx by positively charged liposomes is time and concentration dependent, requiring at least a 5-min incubation with 2.5 mM phosphatidylcholine containing 20% stearylamine for maximum effect. Stimulation is not appreciably reversed by washing the cells. Similar increases are observed for influx and efflux so that there is no change in the steady-state methotrexate electrochemical-potential difference across the cell membrane. The increase influx appears to be a stimulation of the carrier-mediated transport process for methotrexate since both control and stimulated influx are abolished by the competitive inhibitor, 5-formyltetrahydrofolate or the sulfhydryl group inhibitor, p-chloromercuriphenylsulfonic acid and the Q10 of the system remains unchanged. Influx of 5-methyltetrahydrofolate, which shares the same transport carrier as methotrexate, is also stimulated. However, the transport of folic acid, which is structurally similar to methotrexate but does not utilize the carrier, is unaffected. The kinetic change induced by positively charged liposomes is an increase in the Vma in, while the Kt in remains unchanged. Trans-stimulation of methotrexate influx by 5-formyltetrahydrofolate occurs to the same extent in the presence or absence of positively charged liposomes. The liposomes have no apparent effect on the intracellular water, the extracellular space, or the chloride distribution ratio. The data suggest that interaction of positively charged liposomes with Ehrlich ascites tumor cells accelerates the rate of transposition of the membrane carrier system for methotrexate, altering the kinetics of transport without a change in transport thermodynamics.

Animals↗

PubMed1991

Inhibition of mammalian thymidylate synthase by 10-formyltetrahydropteroylpolyglutamate.

Reduced derivatives of 10-formylfolate have been evaluated as inhibitors of mammalian thymidylate synthase (EC 2.1.1.45) from H35 hepatoma cells. With 5,10-methylenetetrahydrofolylheptaglutamate as the substrate, 10-formyltetrahydrofolylmonoglutamate is a competitive inhibitor with a Ki of 2.4 microM, which is reduced to 0.1 microM for the heptaglutamate derivative. 10-Formyldihydrofolylmono- and -heptaglutamate are approximately threefold less inhibitory than the tetrahydro analog. The concentrations of 10-formyltetrahydrofolate and 10-formyldihydrofolate were measured in dividing hepatoma cells by a novel enzymatic assay and were found to be 5 microM and undetectable, respectively. These results suggest that the concentration of 10-formyltetrahydrofolate within the dividing cells has the potential to severely inhibit or modulate thymidylate biosynthesis.

Animals↗

PubMed1982

Time-course studies on the effects of oestradiol administration on the activity of some folate-metabolizing enzymes in chicken liver.

1. The specific activities of some enzymes of folate metabolism were measured in the liver of folate-treated and folate-deficient immature female chickens at intervals up to 9 days after an initial administration of 17 beta-oestradiol dipropionate. 2. An increase in dihydrofolate reductase activity after 3 days and decreases in 10-formyltetrahydrofolate synthetase and 5,10-methylenetetrahydrofolate dehydrogenase activities after 2 days became evident as a result of oestradiol treatment. 3. After 9 days, oestradiol caused a small decrease in glutamate formiminotransferase activity. 4. In folate-deficient chickens the activity of dihydrofolate reductase was decreased and that of serine hydroxymethyltransferase, 10-formyltetrahydrofolate synthetase and 5,10-methylenetetrahydrofolate dehydrogenase was increased in both non-hormone-treated and oestradiol-treated animals.

Animals↗

PubMed2006

Leucovorin-induced resistance against FDH growth suppressor effects occurs through DHFR up-regulation.

10-Formyltetrahydrofolate dehydrogenase (FDH) converts 10-formyltetrahydrofolate to tetrahydrofolate (THF). Expression of the enzyme in FDH-deficient cancer cells induces cytotoxicity that can be reversed by supplementation with high concentrations of a reduced folate, 5-formyl-THF (leucovorin). In contrast, non-tumor cells are resistant to FDH. The present study was undertaken to investigate mechanisms that could protect cells against FDH suppressor effects. Using 10 microM leucovorin supplementation of FDH-sensitive A549 cells transfected for FDH expression, we selected clones that have acquired resistance against FDH. Resistant cells expressed high levels of FDH and were capable of growing after withdrawal of leucovorin. These cells, however, have increased doubling time due to prolonged S phase. They also have significantly increased levels of total folate pool and THF/5,10-methylene-THF pool while the level of 10-formyl-THF was two-fold lower than in parental FDH-sensitive cells. We have shown that the FDH-catalyzed reaction proceeds at about a three-fold slower rate at the ratio of 10-formyl-THF/THF corresponding to the resistant cells than at the ratio corresponding to parental sensitive cells, due to product inhibition (KI is 2.35 microM). FDH-resistant cells have strongly up-regulated dihydrofolate reductase (DHFR) that is proposed to be a mechanism for the alteration of folate pools and a key component of the acquired resistance. Elevation of DHFR in A549 cells by transient transfection decreased sensitivity to FDH toxicity and allowed selection of FDH-resistant clones. DHFR-induced repression of FDH catalysis could be an S phase-related metabolic adjustment that provides protection against FDH suppressor effects.

Base Sequence↗

PubMed2000

Structure of a murine cytoplasmic serine hydroxymethyltransferase quinonoid ternary complex: evidence for asymmetric obligate dimers.

Serine hydroxymethyltransferase (SHMT) is a pyridoxal phosphate-dependent enzyme that catalyzes the reversible conversion of serine and tetrahydrofolate to glycine and methylenetetrahydrofolate. This reaction generates single carbon units for purine, thymidine, and methionine biosynthesis. The enzyme is a homotetramer comprising two obligate dimers and four pyridoxal phosphate-bound active sites. The mammalian enzyme is present in cells in both catalytically active and inactive forms. The inactive form is a ternary complex that results from the binding of glycine and 5-formyltetrahydrofolate polyglutamate, a slow tight-binding inhibitor. The crystal structure of a close analogue of the inactive form of murine cytoplasmic SHMT (cSHMT), lacking only the polyglutamate tail of the inhibitor, has been determined to 2.9 A resolution. This first structure of a ligand-bound mammalian SHMT allows identification of amino acid residues involved in substrate binding and catalysis. It also reveals that the two obligate dimers making up a tetramer are not equivalent; one can be described as "tight-binding" and the other as "loose-binding" for folate. Both active sites of the tight-binding dimer are occupied by 5-formyltetrahydrofolate (5-formylTHF), whose N5-formyl carbon is within 4 A of the glycine alpha-carbon of the glycine-pyridoxal phosphate complex; the complex appears to be primarily in its quinonoid form. In the loose-binding dimer, 5-formylTHF is present in only one of the active sites, and its N5-formyl carbon is 5 A from the glycine alpha-carbon. The pyridoxal phosphates appear to be primarily present as geminal diamine complexes, with bonds to both glycine and the active site lysine. This structure suggests that only two of the four catalytic sites on SHMT are catalytically competent and that the cSHMT-glycine-5-formylTHF ternary complex is an intermediate state analogue of the catalytic complex associated with serine and glycine interconversion.

Animals↗

PubMed2006

Modular organization of FDH: Exploring the basis of hydrolase catalysis.

An abundant enzyme of liver cytosol, 10-formyltetrahydrofolate dehydrogenase (FDH), is an interesting example of a multidomain protein. It consists of two functionally unrelated domains, an aldehyde dehydrogenase-homologous domain and a folate-binding hydrolase domain, which are connected by an approximately 100-residue linker. The amino-terminal hydrolase domain of FDH (Nt-FDH) is a homolog of formyl transferase enzymes that utilize 10-formyl-THF as a formyl donor. Interestingly, the concerted action of all three domains of FDH produces a new catalytic activity, NADP+-dependent oxidation of 10-formyltetrahydrofolate (10-formyl-THF) to THF and CO2. The present studies had two objectives: First, to explore the modular organization of FDH through the production of hybrid enzymes by domain replacement with methionyl-tRNA formyltransferase (FMT), an enzyme homologous to the hydrolase domain of FDH. The second was to explore the molecular basis for the distinct catalytic mechanisms of Nt-FDH and related 10-formyl-THF utilizing enzymes. Our studies revealed that FMT cannot substitute for the hydrolase domain of FDH in order to catalyze the dehydrogenase reaction. It is apparently due to inability of FMT to catalyze the hydrolysis of 10-formyl-THF in the absence of the cosubstrate of the transferase reaction despite the high similarity of the catalytic centers of the two enzymes. Our results further imply that Ile in place of Asn in the FDH hydrolase catalytic center is an important determinant for hydrolase catalysis as opposed to transferase catalysis.

Amino Acid Sequence↗

PubMed1980

Marrow cells from patients with untreated pernicious anaemia cannot use tetrahydrofolate normally.

Folate analogues were added to human bone marrow cells to determine their effect on deoxyuridine utilization in the deoxyuridine suppression test. Formyltetrahydrofolates fully corrected the impairment of dU utilization in pernicious anaemia marrows but tetrahydrofolate was relatively ineffective. All these analogues were effective in megaloblastic marrows from folate deficient patients. Formyltetrahydrofolates also enhanced dU utilization by normal human marrows whereas methyltetrahydrofolate reduced its use. In terms of the methylfolate trap hypothesis, the expectation that cobalamin-deficient marrows would be able to use tetrahydrofolate normally was not realized.

Anemia, Megaloblastic↗

PubMed1999

Folate and folate-dependent enzymes associated with rat CNS development.

Folic acid and its derivatives are important mediators in growth-related cellular processes. The concentration of folate and two folate-dependent enzymes, 10-formyltetrahydrofolate synthetase (10-FTHFS) and 10-formyltetrahydrofolate dehydrogenase (10-FTHFDH), was determined in brain regions over the early period of rat development. Folate concentrations determined at birth were high in all brain regions studied. During the first 2 weeks, folate concentrations declined steadily, followed by a period of significant increase. High and invariant activity of 10-FTHFS was found throughout the period of study. Low amounts of 10-FTHFDH were seen for the first 2 weeks, but increased significantly from postnatal days 14 to 28. These changes correlated with changes determined in the concentration of folate, supporting the idea that this protein is involved with folate uptake and/or storage. Furthermore, immunohistochemical expression of 10-FTHFDH in different rat brain regions revealed glial cells as a preferential cellular location for this folate-binding protein.

Aging↗

PubMed1983

Chronic cobalamin inactivation impairs folate polyglutamate synthesis in the rat.

Nitrous oxide, by inactivating cobalamin in vivo, produces a suitable animal model for cobalamin 'deficiency.' The synthesis of folate polyglutamate with tetrahydrofolate as substrate is severely impaired in the N2O-treated rat, but is normal with formyltetrahydrofolate as substrate. Methionine restores the capacity of the N2O-treated rat to utilize tetrahydrofolate the minimum effective dose being 16 mumol. S-Adenosylmethionine was somewhat less effective than methionine but 5'methylthioadenosine, a product of S-adenosylmethionine metabolism, was significantly more effective than methionine in correcting the defect in folate polyglutamate synthesis. 5'Methylthioadenosine is metabolised to yield formate. It is suggested that these compounds have their effect in correcting folate polyglutamate synthesis by supplying formate for the formylation of tetrahydrofolate. Formyltetrahydrofolate, at least in the cobalamin-inactivated animal, is the required substrate for folate polyglutamate synthesis. Cobalamin is concerned with the maintenance of normal levels of methionine and this in turn is a major source of formate through S-adenosylmethionine and 5'methylthioadenosine.

Adenosine↗

PubMed1987

Studies on the role of folic acid and folate-dependent enzymes in human methanol poisoning.

Methanol toxicity is observed in monkeys and humans but is not seen in rats or mice. The expression of methanol poisoning is related to the ability of an animal to metabolize formate to carbon dioxide. Since the rate of formate oxidation is related to hepatic tetrahydrofolate (H4folate) content and the activities of folate-dependent enzymes, studies were designed to determine hepatic concentrations of H4folate and activities of folate-dependent enzymes of human liver and livers of species considered insensitive to methanol poisoning. An excellent correlation between hepatic H4folate and maximal rates of formate oxidation has been observed. In human liver, H4folate levels were only 50% of those observed for rat liver and similar to those found in monkey liver. Total folate was also lower (60% decreased) in human liver than that found in rat or monkey liver. Interestingly, mouse liver contains much higher hepatic H4folate and total folate than rat or monkey liver. This is consistent with higher formate oxidation rates in this species. A second important observation has been made. 10-Formyltetrahydrofolate dehydrogenase activity, the enzyme catalyzing the final step of formate oxidation to carbon dioxide, was markedly reduced in both monkey and human liver. Thus, two mechanisms may be operative in explaining low formate oxidation in species susceptible to methanol toxicity, low hepatic H4folate levels and reduced hepatic 10-formyltetrahydrofolate dehydrogenase activity.

Acidosis↗

PubMed1989

Toxicity and antitumor activity of 5-fluorouracil in combination with leucovorin. Role of dose schedule and route of administration of leucovorin.

Clinically, 5-formyltetrahydrofolate (leucovorin, folinic acid, LV) in combination with 5-fluorouracil (5-FU) has been used at various doses, schedules, and routes of administration with therapeutic benefit to patients with advanced colorectal carcinoma and breast carcinoma. Clinical experiences have been primarily with LV doses of 25, 200, and 500 mg/m2 administered by either short-term intravenous infusion, daily continuous infusion, or orally. In patients with lung carcinoma, oral administration of dl-LV at 125 mg/m2 hourly for 4 hours (total dose of 500 mg/m2) gave the following peak plasma folate concentrations: dl-LV, 4.6 + 1.9 microM; 5-methyltetrahydrofolate, 4.3 + 2.1 microM; and no detectable l-LV in most cases. The d-LV/5-methyltetrahydrofolate ratios, however, were lower for the oral route than for the same dl-LV dose administered by 2-hour intravenous infusion in the same patient. To determine if there is a relationship between the dose, schedule, or route of administration and the therapeutic efficacy of LV combined with 5-FU, studies were carried out in rats with transplantable colon carcinoma. 5-Formyltetrahydrofolate was administered at various doses by either 2-hour infusion, 2-day continuous intravenous infusion, or by divided hourly oral doses for 4 hours. In all cases, the total doses of dl-LV administered were 100, 200, and 400 mg/kg. Data obtained to date indicate: (1) plasma folate concentrations by intravenous administration were dose dependent, but lower and saturable concentrations of folates were observed by oral administration; and (2) while the concentrations of 5-methyltetrahydrofolate achieved by the 2-hour infusion schedule were relatively constant and independent of the dose of dl-LV administered, conversion of dl-LV to 5-methyltetrahydrofolate with the 2-day infusion of 100, 200, and 400 mg/kg was dose dependent. In humans, however, conversion was independent of the route of administration of dl-LV (19% for the 2-hour infusion and 23% for the 5-day infusion of 500 mg/m2 dose). Preliminary results for antitumor activity of 5-FU in combination with dl-LV, administered by either 2-hour intravenous infusion (400 mg/kg/day for 5 days) or orally (100 mg/kg/hour for 4 hours), yielded similar inhibition of in vivo tumor growth, each being greater than what was achieved with 5-FU alone.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed2005

Structural and functional characterization of a 5,10-methenyltetrahydrofolate synthetase from Mycoplasma pneumoniae (GI: 13508087).

Mycoplasma pneumoniae 5,10-methenyltetrahydrofolate synthetase [MTHFS; also known as 5-formyltetrahydrofolate cycloligase; Enzyme Commission (EC) 6.3.3.2] belongs to a large cycloligase protein family with 97 sequence homologues from bacteria to human. To help define the molecular (biochemical and biophysical) function of the M. pneumoniae MTHFS, we have previously determined its crystal structure at 2.2 A resolution (Chen et al., Proteins 2004;56:839-843). In this current study, activity assays confirmed the functionality of the recombinant protein, with K(m) = 165 microM for 5-formyltetrahydrofolate (5-FTHF) and K(m) = 166 microM for MgATP. The methenyltetrahydrofolate activity of M. pneumoniae MTHFS has a requirement for divalent metal ions with Mg2+ being most effective, and an absolute requirement for nucleoside 5'-triphosphates with adenosine triphosphate (ATP) being most effective. Crystallization in the presence of substrates (MgATP, with or without 5-FTHF) produced the complex structures of the protein with adenosine diphosphate (ADP) and phosphate at 2.2 A resolution; with ADP, phosphate, and 5-FTHF at 2.5 A resolution. These structures directly demonstrated that the role of Mg2+ in the reaction is to form the ATP--Mg2+-enzyme complex.

Adenosine Diphosphate↗

PubMed2001

Disruption of histidine catabolism in NEUT2 mice.

Homozygous NEUT2 mice lack cytosolic 10-formyltetrahydrofolate dehydrogenase (FDH; Champion et al. (1994) Proc. Natl. Acad. Sci. USA 91, 11,338-11,342) and as a consequence should be unable to oxidize carbon 2 of l-histidine to CO2 via 10-formyltetrahydrofolate in liver cytosol. There was essentially no oxidation of l-[2-14C]histidine to 14CO2 in homozygous NEUT2 mice, but 52% of the [2-14C]l-histidine dose was recovered in the urine within 24 h. Analysis of urine samples for [14C]formiminoglutamate, the expected excretion product, was negative; however, [14C]urocanic acid was detected. Investigation of histidine catabolism via the folate-dependent deamination pathway revealed no detectable urocanase activity in homozygous NEUT2 mice, while heterozygous NEUT2 mice had 50% urocanase activity compared to normal mice. Histidase and formiminotransferase-cyclodeaminase, also on the histidine deamination pathway, had similar specific activities in normal and NEUT2 mice. Histidine-pyruvate aminotransferase, the first enzyme of the alternate histidine transamination catabolic pathway did not appear to be affected by the loss of urocanase. Based on the excretion of urocanic acid it is estimated that NEUT2 mice catabolize approximately 40 micromol/day via the deamination pathway. The loss of urocanase activity in homozygous NEUT2 mice may allow these mice to survive the disruption in folate metabolism by sparing the liver cytosolic tetrahydrofolate pool.

Animals↗

PubMed1994

Disposition of leucovorin and its metabolites in the plasma, intestinal epithelium, and intraperitoneal L1210 cells of methotrexate-pretreated mice.

Leucovorin (LV or 5-CHOFH4) has had long-standing clinical use as a rescue agent from the systemic toxic effects of methotrexate (MTX). Because the mouse has been the animal model most used to investigate MTX therapy, direct tissue assessment of LV and its reduced-folate metabolites was undertaken in the plasma, intestinal epithelium, and intraperitoneal L1210 cells of MTX-pretreated mice using a ternary-complex-based assay method. The results show that total folate accumulation and depletion in tissues is closely related to plasma levels, with somewhat greater persistence occurring in tissues, presumably due to polyglutamylation. Examination of individual folates in plasma showed that the combined 5,10-methylenetetrahydrofolate (CH2FH4) plus tetrahydrofolate (FH4) pool was the most extensively elevated pool other than that of the parent compound [S]-5-formyltetrahydrofolate ([S]-5-CHOFH4). The dihydrofolate (FH2) also became elevated, whereas the 5-methyltetrahydrofolate (5-CH3FH4) remained unchanged. Individual folates that were elevated in tissues were generally the same as those elevated in plasma, the exception being a significant accumulation of 10-formyltetrahydrofolate (10-CHOFH4) in both intestinal epithelial and L1210 cells. The elevation of FH2 in L1210 cells was greater and persisted longer than that in intestinal epithelium, whereas the opposite was true for CH2FH4 + FH4. This differential effect in tumor versus epithelial tissue is consistent with the selective rescue of normal tissue by LV.

Animals↗

PubMed2003

Specific and sensitive quantification of folate vitamers in foods by stable isotope dilution assays using high-performance liquid chromatography-tandem mass spectrometry.

Stable isotope dilution assays were developed for the quantification of the folate vitamers 5-methyltetrahydrofolate, 5-formyltetrahydrofolate, tetrahydrofolate, 10-formylfolate and pteroylglutamic acid in food samples by using deuterated isotopomers as internal standards. Vitamers and their labeled analogues were analyzed simultaneously by HPLC/MS/MS using selected reaction monitoring, which allowed a higher specificity than other methods published previously. Sample preparation involved treatment by protease in sequence with alpha-amylase and rat serum deconjugase, followed by anion exchange chromatography. The detection limits for 5-methyltetrahydrofolate, 5-formyltetrahydrofolate, tetrahydrofolate, 10-formylfolate and pteroylglutamic acid were found to be 0.5, 1.2, 1.5, 0.6 and 2.6 microg/100 g fresh weight, respectively. Using the new method, folate contents were determined in meat, cereals, and vegetables. Data were in good agreement with literature data, except results for broccoli, which were much lower than reported in previous studies.

Animals↗