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PubMed1998

A mutated murine reduced folate carrier (RFC1) with increased affinity for folic acid, decreased affinity for methotrexate, and an obligatory anion requirement for transport function.

In an ongoing study of structure-function relationships of the murine reduced folate carrier 1 (RFC1), a glutamate to lysine mutation at amino acid 45 was identified in a methotrexate (MTX)-resistant L1210 clonal variant in which MTX and 5-formyltetrahydrofolate (5-CHO-THF) influx was markedly decreased. The characteristics of the mutated carrier, RFC1-E45K, were studied by cDNA transfection into the murine MTXrA line in which endogenous carrier is not functional. Folic acid influx doubled in the transfectant MTXrA-E45K as compared with L1210 or MTXrA cells; in contrast, MTX and 5-CHO-THF influx was only 14 and 27% that of L1210 cells, respectively. 5-CHO-THF influx in MTXrA-E45K cells was characterized by a 12- and 3.6-fold decrease in influx Vmax and Kt respectively, relative to L1210 cells. The folic acid influx Ki in L1210 cells was more than 50-fold greater than that of MTX based upon inhibition of 5-CHO-THF influx. In comparison, the mutated carrier had comparable affinities for folic acid and MTX in MTXrA-E45K cells due to a 7-fold decrease in the folic acid influx Ki and 7-fold increase in the MTX influx Ki. Transport via native RFC1 is inhibited by a variety of anions in L1210 cells associated with an increase in influx Kt. However, influx of 5-CHO-THF in MTXrA-E45K cells in a HEPES buffer (9 mM chloride) was decreased by 70% due to a 3-fold fall in the Vmax. In the complete absence of chloride (K+-HEPES-sucrose buffer) 5-CHO-THF influx was only 10% that in HBS buffer. 5-CHO-THF influx was restored by addition of chloride, fluoride, or nitrate but not by sulfate, phosphate, or ATP which were all inhibitory over a broad range of concentrations. The data suggest that substitution of a positive for a negative amino acid at position 45 results in the loss of RFC1 mobility in the absence of small inorganic anions that bind to, and neutralize the positive charge on, the lysine residue. Inhibition by higher charged anions may be due to interactions at another carrier site present in both the mutated and wild type carrier. This and other studies suggest that amino acids in the first predicted transmembrane domain play an important role in determining the spectrum of affinities for, and mobility of, RFC1 and is a cluster region for mutations when cells are placed under selective pressure with antifolates that utilize RFC1 as the major route of entry into mammalian cells.

Amino Acid Substitution↗
PubMed1998

Mutations in the reduced folate carrier gene which confer dominant resistance to 5,10-dideazatetrahydrofolate.

L1210/D3 mouse leukemia cells are resistant to 5, 10-dideazatetrahydrofolate due to expansion of cellular folate pools which block polyglutamation of the drug (Tse, A., and Moran, R. G. (1998) J. Biol. Chem. 273, 25944-25952). These cells were found to have two point mutations in the reduced folate carrier (RFC), resulting in a replacement of isoleucine 48 by phenylalanine and of tryptophan 105 by glycine. Each mutation contributes to the resistance phenotype. Genomic DNA from resistant cells contained both the wild-type and mutant alleles, but wild-type message was not detected. Folic acid was a much better substrate, and 5-formyltetrahydrofolate was a poorer substrate for transport in L1210/D3 cells relative to L1210 cells. Enhanced transport of folic acid was due to a marked, approximately 20-fold, decrease in the influx Km. Influx of methotrexate and 5,10-dideazatetrahydrofolate were minimally altered. Transfection of mutated rfc cDNA into RFC-null L1210/A cells produced the substrate specificity and 5, 10-dideazatetrahydrofolate resistance observed in the L1210/D3 line. Transfection of the mutant cDNA into wild-type cells also conferred resistance to 5,10-dideazatetrahydrofolate. We conclude that the I48F and W105G mutations in RFC caused resistance to 5, 10-dideazatetrahydrofolate, that the region of the RFC protein near these two positions defines the substrate-binding site, that the wild-type allele was silenced during the multistep development of resistance, and that this mutant phenotype represents a genetically dominant trait.

Animals↗
PubMed2000

Purification and properties of a folate-catabolizing enzyme.

We have identified and purified to homogeneity an enzyme from rat liver that catalyzes the oxidative catabolism of 5-formyltetrahydrofolate to p-aminobenzoylglutamate and a pterin derivative. Purification of the enzyme utilized six column matrices, including a pterin-6-carboxylic acid affinity column. Treatment of crude rat liver extracts with EDTA or heat decreased the specific activity of the enzyme by up to 85%. Peptides generated from the purified protein were sequenced and found to be identical to primary sequences present within rat light chain or heavy chain ferritin. Commercial rat ferritin did not display catabolic activity, but activity could be acquired with iron loading. The purified enzyme contained 2000 atoms of iron/ferritin 24-mer and displayed similar electrophoretic properties as commercial rat liver ferritin. The ferritin-catalyzed reaction displayed burst kinetics, and the enzyme catalyzed only a single turnover in vitro. Expression of rat heavy chain ferritin cDNA resulted in increased rates of folate turnover in cultured Chinese hamster ovary cells and human mammary carcinoma cells and reduced intracellular folate concentrations in Chinese hamster ovary cells. These results indicate that ferritin catalyzes folate turnover in vitro and in vivo and may be an important factor in regulating intracellular folate concentrations.

Amino Acid Sequence↗
PubMed2001

Oxidative decarboxylation of UDP-glucuronic acid in extracts of polymyxin-resistant Escherichia coli. Origin of lipid a species modified with 4-amino-4-deoxy-L-arabinose.

Addition of the 4-amino-4-deoxy-l-arabinose (l-Ara4N) moiety to the phosphate groups of lipid A is implicated in bacterial resistance to polymyxin and cationic antimicrobial peptides of the innate immune system. The sequences of the products of the Salmonella typhimurium pmrE and pmrF loci, both of which are required for polymyxin resistance, recently led us to propose a pathway for l-Ara4N biosynthesis from UDP-glucuronic acid (Zhou, Z., Lin, S., Cotter, R. J., and Raetz, C. R. H. (1999) J. Biol. Chem. 274, 18503-18514). We now report that extracts of a polymyxin-resistant mutant of Escherichia coli catalyze the C-4" oxidation and C-6" decarboxylation of [alpha-(32)P]UDP-glucuronic acid, followed by transamination to generate [alpha-(32)P]UDP-l-Ara4N, when NAD and glutamate are added as co-substrates. In addition, the [alpha-(32)P]UDP-l-Ara4N is formylated when N-10-formyltetrahydrofolate is included. These activities are consistent with the proposed functions of two of the gene products (PmrI and PmrH) of the pmrF operon. PmrI (renamed ArnA) was overexpressed using a T7 construct, and shown by itself to catalyze the unprecedented oxidative decarboxylation of UDP-glucuronic acid to form uridine 5'-(beta-l-threo-pentapyranosyl-4"-ulose diphosphate). A 6-mg sample of the latter was purified, and its structure was validated by NMR studies as the hydrate of the 4" ketone. ArnA resembles UDP-galactose epimerase, dTDP-glucose-4,6-dehydratase, and UDP-xylose synthase in oxidizing the C-4" position of its substrate, but differs in that it releases the NADH product.

Amino Sugars↗
PubMed2002

Cloning and characterization of methenyltetrahydrofolate synthetase from Saccharomyces cerevisiae.

The folate derivative 5-formyltetrahydrofolate (folinic acid; 5-CHO-THF) was discovered over 40 years ago, but its role in metabolism remains poorly understood. Only one enzyme is known that utilizes 5-CHO-THF as a substrate: 5,10-methenyltetrahydrofolate synthetase (MTHFS). A BLAST search of the yeast genome using the human MTHFS sequence revealed a 211-amino acid open reading frame (YER183c) with significant homology. The yeast enzyme was expressed in Escherichia coli, and the purified recombinant enzyme exhibited kinetics similar to previously purified MTHFS. No new phenotype was observed in strains disrupted at MTHFS or in strains additionally disrupted at the genes encoding one or both serine hydroxymethyltransferases (SHMT) or at the genes encoding one or both methylenetetrahydrofolate reductases. However, when the MTHFS gene was disrupted in a strain lacking the de novo folate biosynthesis pathway, folinic acid (5-CHO-THF) could no longer support the folate requirement. We have thus named the yeast gene encoding methenyltetrahydrofolate synthetase FAU1 (folinic acid utilization). Disruption of the FAU1 gene in a strain lacking both 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) transformylase isozymes (ADE16 and ADE17) resulted in a growth deficiency that was alleviated by methionine. Genetic analysis suggested that intracellular accumulation of the purine intermediate AICAR interferes with a step in methionine biosynthesis. Intracellular levels of 5-CHO-THF were determined in yeast disrupted at FAU1 and other genes encoding folate-dependent enzymes. In fau1 disruptants, 5-CHO-THF was elevated 4-fold over wild-type yeast. In yeast lacking MTHFS along with both AICAR transformylases, 5-CHO-THF was elevated 12-fold over wild type. 5-CHO-THF was undetectable in strains lacking SHMT activity, confirming SHMT as the in vivo source of 5-CHO-THF. Taken together, these results indicate that S. cerevisiae harbors a single, nonessential, MTHFS activity. Growth phenotypes of multiply disrupted strains are consistent with a regulatory role for 5-CHO-THF in one-carbon metabolism and additionally suggest a metabolic interaction between the purine and methionine pathways.

Amino Acid Sequence↗
PubMed2003

Pyridoxal phosphate inhibits dynamic subunit interchange among serine hydroxymethyltransferase tetramers.

Cytoplasmic serine hydroxymethyltransferase (cSHMT) is a tetrameric, pyridoxal phosphate (PLP)-dependent enzyme that catalyzes the reversible interconversion of serine and tetrahydrofolate to glycine and methylenetetrahydrofolate. The enzyme has four active sites and is best described as a dimer of obligate dimers. Each monomeric subunit within the obligate dimer contributes catalytically important amino acid residues to both active sites. To investigate the interchange of subunits among cSHMT tetramers, a dominant-negative human cSHMT enzyme (DNcSHMT) was engineered by making three amino acid substitutions: K257Q, Y82A, and Y83F. Purified recombinant DNcSHMT protein was catalytically inactive and did not bind 5-formyltetrahydrofolate. Coexpression of the cSHMT and DNcSHMT proteins in bacteria resulted in the formation of heterotetramers with a cSHMT/DNcSHMT subunit ratio of 1. Characterization of the cSHMT/DNcSHMT heterotetramers indicates that DNcSHMT and cSHMT monomers randomly associate to form tetramers and that cSHMT/DNcSHMT obligate dimers are catalytically inactive. Incubation of recombinant cSHMT protein with recombinant DNcSHMT protein did not result in the formation of hetero-oligomers, indicating that cSHMT subunits do not exchange once the tetramer is assembled. However, removal of the active site PLP cofactor does permit exchange of obligate dimers among preformed cSHMT and DNcSHMT tetramers, and the formation of heterotetramers from cSHMT and DNcSHMT homodimers does not affect the activity of the cSHMT homodimers. The results of these studies demonstrate that PLP inhibits dimer exchange among cSHMT tetramers and suggests that cellular PLP concentrations may influence the stability of cSHMT protein in vivo.

Binding Sites↗
PubMed2005

A formyltransferase required for polymyxin resistance in Escherichia coli and the modification of lipid A with 4-Amino-4-deoxy-L-arabinose. Identification and function oF UDP-4-deoxy-4-formamido-L-arabinose.

Modification of the phosphate groups of lipid A with 4-amino-4-deoxy-L-arabinose (L-Ara4N) is required for resistance to polymyxin and cationic antimicrobial peptides in Escherichia coli and Salmonella typhimurium. We previously demonstrated that the enzyme ArnA catalyzes the NAD+-dependent oxidative decarboxylation of UDP-glucuronic acid to yield the UDP-4''-ketopentose, uridine 5'-diphospho-beta-(L-threo-pentapyranosyl-4''-ulose), which is converted by ArnB to UDP-beta-(L-Ara4N). E. coli ArnA is a bi-functional enzyme with a molecular mass of approximately 74 kDa. The oxidative decarboxylation of UDP-glucuronic acid is catalyzed by the 345-residue C-terminal domain of ArnA. The latter shows sequence similarity to enzymes that oxidize the C-4'' position of sugar nucleotides, like UDP-galactose epimerase, dTDP-glucose-4,6-dehydratase, and UDP-xylose synthase. We now show that the 304-residue N-terminal domain catalyzes the N-10-formyltetrahydrofolate-dependent formylation of the 4''-amine of UDP-L-Ara4N, generating the novel sugar nucleotide, uridine 5'-diphospho-beta-(4-deoxy-4-formamido-L-arabinose). The N-terminal domain is highly homologous to methionyl-tRNA(f)Met formyltransferase. The structure of the formylated sugar nucleotide generated in vitro by ArnA was validated by 1H and 13C NMR spectroscopy. The two domains of ArnA were expressed independently as active proteins in E. coli. Both were required for maintenance of polymyxin resistance and L-Ara4N modification of lipid A. We conclude that N-formylation of UDP-L-Ara4N is an obligatory step in the biosynthesis of L-Ara4N-modified lipid A in polymyxin-resistant mutants. We further demonstrate that only the formylated sugar nucleotide is converted in vitro to an undecaprenyl phosphate-linked form by the enzyme ArnC. Because the L-Ara4N unit attached to lipid A is not derivatized with a formyl group, we postulate the existence of a deformylase, acting later in the pathway.

Amino Sugars↗
PubMed2005

Regulation of de novo purine biosynthesis by methenyltetrahydrofolate synthetase in neuroblastoma.

5-Formyltetrahydrofolate (5-formylTHF) is the only folate derivative that does not serve as a cofactor in folate-dependent one-carbon metabolism. Two metabolic roles have been ascribed to this folate derivative. It has been proposed to 1) serve as a storage form of folate because it is chemically stable and accumulates in seeds and spores and 2) regulate folate-dependent one-carbon metabolism by inhibiting folate-dependent enzymes, specifically targeting folate-dependent de novo purine biosynthesis. Methenyltetrahydrofolate synthetase (MTHFS) is the only enzyme that metabolizes 5-formylTHF and catalyzes its ATP-dependent conversion to 5,10-methenylTHF. This reaction determines intracellular 5-formylTHF concentrations and converts 5-formylTHF into an enzyme cofactor. The regulation and metabolic role of MTHFS in one-carbon metabolism was investigated in vitro and in human neuroblastoma cells. Steady-state kinetic studies revealed that 10-formylTHF, which exists in chemical equilibrium with 5,10-methenylTHF, acts as a tight binding inhibitor of mouse MTHFS. [6R]-10-formylTHF inhibited MTHFS with a K(i) of 150 nM, and [6R,S]-10-formylTHF triglutamate inhibited MTHFS with a K(i) of 30 nm. MTHFS is the first identified 10-formylTHF tight-binding protein. Isotope tracer studies in neuroblastoma demonstrate that MTHFS enhances de novo purine biosynthesis, indicating that MTHFS-bound 10-formylTHF facilitates de novo purine biosynthesis. Feedback metabolic regulation of MTHFS by 10-formylTHF indicates that 5-formylTHF can only accumulate in the presence of 10-formylTHF, providing the first evidence that 5-formylTHF is a storage form of excess formylated folates in mammalian cells. The sequestration of 10-formylTHF by MTHFS may explain why de novo purine biosynthesis is protected from common disruptions in the folate-dependent one-carbon network.

Animals↗
PubMed1990

Biochemical rationale for the 5-fluorouracil leucovorin combination and update of clinical experience.

Although 5-fluorouracil has been the drug of choice for the treatment of patients with advanced adenocarcinoma of the colon, the reported response rate dose not exceed 20% with a median survival time less than 9 months. Mechanisms of sensitivity to this agent include: 1) inhibition of thymidylate synthase by FdUMP, the active metabolite of 5-fluorouracil; 2) incorporation of FUTP into cellular RNA; and 3) incorporation of FdUTP into cellular DNA. Recent laboratory preclinical results suggest that the major site of action of 5-fluorouracil when combined with 5-formyltetrahydrofolate (folinic acid, leucovorin) is thymidylate synthase resulting in pronounced and prolonged inhibition of DNA synthesis. This effect is primarily due to the stabilization of FdUMP binding to thymidylate synthase by the reduced folate cofactor, N5, N10-methylenetetrahydrofolate. In this paper, the rationale for the modulation of 5-fluorouracil by leucovorin, the clinical pharmacology and the clinical experience of this combination will be reviewed.

Animals↗
PubMed1990

Biochemical modulation of fluoropyrimidines by antifolates and folates in an in vitro model of human leukemia.

Although 5-fluorouracil (FUra) is one of the most effective cytotoxic agents in the treatment of various solid tumors (carcinomas of the gastro-intestinal tract, breast, head and neck), remissions occur in only 20 to 30% of cases and usually are of short duration. Recently, preclinical studies have shown that the antitumor activity of FUra can be potentiated by modulating the metabolism of this drug by using other substances, in particular antifolates of folates. Pretreatment with antifolates may, by blocking de novo purine biosynthesis and consequently increasing phosphoribosyl pyrophosphate (PRPP) pools, enhance the conversion of FUra to active fluoronucleotide pools via orotate phosphoribosyltransferase. Methotrexate (MTX) pretreatment may also enhance binding of the fluoropyrimidine inhibitor, 5-fluodeoxyuridylate (FdUMP), to the target enzyme, thymidylate synthase (TS), indirectly by increasing dihydrofolate polyglutamates or directly, as MTX polyglutamates, by enhancing the formation of ternary complexes with FdUMP and TS. Exogenous folates, in particular 5-formyltetrahydrofolate (folinate, leucovorin, LV), can, by raising the intracellular levels of 5, 10-methylenetetrahydrofolate, lead to increased formation and stabilization of the ternary complex formed by TS, the folate coenzyme, and FdUMP. In vitro studies have also shown potentiation of FUra cytotoxicity by antifolates and folates against human lymphoblastic leukemia cell lines. Thus, while FUra may have little or no single agent activity in leukemias and lymphomas, it may be converted to an active drug in these neoplasms by appropriate modulation. Clinical studies of sequential MTX-FUra or combined LV-FUra based upon experimental tumor results reviewed herein, are warranted.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed2004

Characterization of the Saccharomyces cerevisiae Fol1 protein: starvation for C1 carrier induces pseudohyphal growth.

Tetrahydrofolate (vitamin B9) and its folate derivatives are essential cofactors in one-carbon (C1) transfer reactions and absolutely required for the synthesis of a variety of different compounds including methionine and purines. Most plants, microbial eukaryotes, and prokaryotes synthesize folate de novo. We have characterized an important enzyme in this pathway, the Saccharomyces cerevisiae FOL1 gene. Expression of the budding yeast gene FOL1 in Escherichia coli identified the folate biosynthetic enzyme activities dihydroneopterin aldolase (DHNA), 7,8-dihydro-6-hydroxymethylpterin-pyrophosphokinase (HPPK), and dihydropteroate synthase (DHPS). All three enzyme activities were also detected in wild-type yeast strains, whereas fol1Delta deletion strains only showed background activities, thus demonstrating that Fol1p catalyzes three sequential steps of the tetrahydrofolate biosynthetic pathway and thus is the central enzyme of this pathway, which starting from GTP consists of seven enzymatic reactions in total. Fol1p is exclusively localized to mitochondria as shown by fluorescence microscopy and immune electronmicroscopy. FOL1 is an essential gene and the nongrowth phenotype of the fol1 deletion leads to a recessive auxotrophy for folinic acid (5'-formyltetrahydrofolate). Growth of the fol1Delta deletion strain on folinic acid-supplemented rich media induced a dimorphic switch with haploid invasive and filamentous pseudohyphal growth in the presence of glucose and ammonium, which are known suppressors of filamentous and invasive growth. The invasive growth phenotype induced by the depletion of C1 carrier is dependent on the transcription factor Ste12p and the flocullin/adhesin Flo11p, whereas the filamentation phenotype is independent of Ste12p, Tec1p, Phd1p, and Flo11p, suggesting other signaling pathways as well as other adhesion proteins.

Aldehyde-Lyases↗
PubMed1993

5-Methyltetrahydrofolate transport in basolateral membrane vesicles from human liver.

Transport of 5-methyltetrahydrofolate was studied in vesicles isolated from the basolateral membrane (BLM) of human liver. Uptake was mostly via transport into an osmotically active intravesicular space, with some membrane binding (approximately 20%). Transport was more rapid with an imposed pH gradient (pHout = 5.0, pHin = 7.5) as compared with either pHout = pHin = 5.0 or pHout = pHin = 7.5. Transport under the influence of a pH gradient showed a transient overshoot; uptake at 60 s was 4.2 times higher than at equilibrium (60 min). Transport in the presence of a pH gradient was saturable; apparent Km = 0.55 mumol/L and Vmax = 1.98 nmol.g protein-1.10 s-1. Transport was not saturable at pHout = pHin = 7.5. Transport was inhibited by the structural analogs 5-formyltetrahydrofolate, folic acid, and methotrexate, and was electroneutral in nature. These results suggest that 5-methyltetrahydrofolate transport in human BLM vesicles is via carrier-mediated cotransport with hydrogen ions.

Biological Transport↗
PubMed2006

Chronic cigarette smoking is associated with diminished folate status, altered folate form distribution, and increased genetic damage in the buccal mucosa of healthy adults.

BACKGROUND: Smoking causes genetic damage in buccal cells and increases the risk of oral cancer. Because folate is instrumental in DNA synthesis and repair, it is a determinant of genetic stability and therefore might attenuate the genotoxic effects of smoking. OBJECTIVE: Our aim was to compare the presence of folate metabolites and select indicators of genetic damage in the mouths of chronic smokers and nonsmokers. DESIGN: Dietary, biochemical, and molecular correlates of folate status were measured in healthy smoker (n = 35) and nonsmoker (n = 21) groups of comparable age, sex, and body mass indexes. RESULTS: After correction for dietary intake, the smokers displayed lower plasma, erythrocyte, and buccal mucosal cell (BMC) folate (20%, 32%, and 50% lower, respectively; P < 0.05) and lower plasma vitamin B-12 and pyridoxal 5-phosphate (P < 0.05) than did nonsmokers. Folate in the BMCs of smokers comprised significantly greater proportions of pteroylmonoglutamate, formyltetrahydrofolate, and 5,10-methenyltetrahyrofolate than did folate in the BMCs of nonsmokers. Although the degree of genomic methylation and uracil incorporation in the buccal cells of the 2 groups were not significantly different, the BMC micronucleus index, a cytologic indicator of genetic damage, in the smokers was 2-fold that of the nonsmokers (9.57 compared with 4.44 micronuclei/1000 cells; P < 0.0001). Neither systemic nor oral folate status was an independent predictor of micronuclei. CONCLUSIONS: Chronic smoking is associated with a lower systemic status of several B vitamins, reduced oral folate, and changes in folate form distribution in the mouth. However, the cytologic damage that is evident in the mouths of smokers does not correlate with oral folate status.

Adult↗
PubMed1984

Fluorometric determination of folacin in biological materials using high performance liquid chromatography.

A procedure was developed for the quantitation of the major folacin compounds present in foods and other biological materials. Extraction conditions were selected to provide conversion of 10-formyltetrahydrofolic acid (10-HCO-H4folic acid) to 5-HCO-H4folic acid. Polyglutamyl folates were deconjugated with hog kidney conjugase. The resulting folacin monoglutamates were separated by reverse-phase high performance liquid chromatography after extract purification on an anion exchange column. Detection of H4folic acid and its substituted derivatives was performed by monitoring the native fluorescence of the reduced folates in the acidic mobile phase. Folic acid, H2folic acid and also H4folic acid were measured fluorometrically by using an oxidative postcolumn derivatization system in series with the first fluorometer. Detection limits ranged from 0.03 to 2.3 pmol/100 microliters injection for the various folates. The validity of the method was supported by recovery and fluorescence spectral studies and by comparison with Lactobacillus casei assays for a variety of samples. Analysis of rat liver following a single pulse dose of 3H-labeled folic acid indicated large differences between the patterns of radiolabeled and endogenous folates.

Animals↗
PubMed1995

Simultaneous measurement of one-carbon and polyglutamate derivatives of folic acid in rat liver using enzymatic interconversions of folates followed by ternary complex formation with thymidylate synthetase and 5-fluorodeoxyuridylic acid: standardization of the method.

A sensitive assay is needed for the measurement of individual folate derivatives in samples that contain low concentrations of folate. The ternary complex method for the determination of folylpolyglutamates has been combined with procedures for interconverting folate derivatives to provide a method capable of measuring 28 different folate derivatives in biological samples. The method takes advantage of the properties of the ternary complex formed with thymidylate synthetase, fluorodeoxyuridylic acid and 5,10-methylenetetrahydrofolic acid. In the presence of excess purified thymidylate synthetase and excess [3H]fluorodeoxyuridylic acid, limiting concentrations of folates were converted to 5,10-methylenetetrahydrofolate using purified folate interconverting enzymes. This process separated folate derivatives into four groups: Tetrahydrofolate + 5,10-methylenetetrahydrofolate; dihydrofolate; 5,10-methenyl-, 5-formyl-, and 10-formyltetrahydrofolates and 5-methyltetrahydrofolate. Within groups specificity was good, showing little overlap between folates. Sensitivities to 100 fmol of folate were achievable and to 1 pmol were standard. Recoveries were linear for each of the groups in this system to 50 pmol of folate. Ternary complexes containing different polyglutamates were separated by isoelectric focusing, visualized by fluorography and measured by densitometry. The densitometry was linear with folate concentration in the range 20-200 fmol for each of the polyglutamates. Primary and secondary coefficients of variation were determined. This method provides the sensitivity to measure individual folates in the femtomole range and the flexibility to determine the concentrations of 28 separate pools of folate derivatives, differentiating between derivatives of the pteridine moiety and glutamate chain length simultaneously.

Animals↗
PubMed1990

Clinical pharmacokinetics of high-dose leucovorin calcium after intravenous and oral administration.

The clinical formulation of leucovorin calcium (leucovorin, LV) is a mixture of stereoisomers [(6R,S)-5-formyltetrahydrofolate], which have been shown to differ significantly in plasma clearance and route of elimination after intravenous administration; the (6S) isomer is rapidly converted to 5-CH3 tetrahydrofolate (5-CH3 THF), and the (6R) isomer is slowly eliminated by renal excretion. The relative importance of (6S) LV and 5-CH3 THF in expanding reduced folate pools in tumor cells is unknown, but it is known that high concentrations of (6R) LV can support growth of folate-depleted cells and thus have the potential to interfere with the biological activity of the (6S) isomer. To examine the pharmacokinetics of the LV isomers and metabolites, we administered 1,000 mg of LV to five normal subjects as a 2-hour intravenous infusion and in divided oral 100-mg doses given over 24 hours. Plasma and urine samples were analyzed by reverse phase followed by chiral high-performance liquid chromatography. Following intravenous administration, peak plasma concentrations of (6R) LV, (6S) LV, and 5-CH3 THF were 148 +/- 32, 59.1 +/- 22, and 17.8 +/- 17 microM, respectively. During oral administration of LV, virtually no (6S) LV appeared in the plasma. Steady-state plasma concentrations of (6R) LV and 5-CH3 THF were approximately 1.5 +/- 0.23 and 2.8 +/- 0.41 microM, respectively. Intravenous administration of LV resulted in an area under the curve (AUC) for (6R) LV that was more than four times that of the biologically active (6S) folates, whereas oral administration produced an AUC for (6S) reduced folates [(6S) LV and 5-CH3 THF] that was approximately twice that of (6R) LV. After administration of high doses of LV intravenously, conversion of (6S) LV to 5-CH3 THF was saturable, as indicated by the prolonged (6S) LV half-life of 58 minutes and the slow (6S) LV clearance of 119.2 +/- 38 mL/min, compared with previously reported data for administration of low doses. This study illustrates that intravenous administration of LV produces equivalent AUCs of (6S) LV and 5-CH3 THF but a substantially higher AUC for (6R) LV. Oral administration over 24 hours results in an AUC of 5-CH3 THF equivalent to that obtained after intravenous dosing in the presence of only small amounts of (6R) LV. The optimal route of LV administration will ultimately be determined by ongoing studies of the cellular pharmacology of LV that will determine if high concentrations of (6R) LV interfere with the biological activity of the (6S) reduced folates.

Administration, Oral↗
PubMed2005

A polymorphism in the MTHFD1 gene increases a mother's risk of having an unexplained second trimester pregnancy loss.

Low maternal folate or vitamin B12 status has been implicated in numerous pregnancy complications including spontaneous abortion. The primary aim of this study was to test a polymorphism within the trifunctional folate enzyme MTHFD1 (5,10-methylenetetrahydrofolate dehydrogenase, 5,10-methenyltetrahydrofolate cyclohydrolase, 10-formyltetrahydrofolate synthetase) for an association with a mother's risk of having an unexplained second trimester pregnancy loss. We genotyped 125 women who had at least one unexplained spontaneous abortion or intrauterine fetal death between 13 and 26 weeks gestation and 625 control women with no history of prior pregnancy loss. Our study is the first to identify an association between the MTHFD1 1958G-->A (R653Q) polymorphism and the maternal risk of having an unexplained second trimester pregnancy loss. Women who are MTHFD1 1958AA homozygous have a 1.64-fold increased risk of having an unexplained second trimester loss compared to women who are MTHFD1 1958AG or 1958GG [OR 1.64 (1.05-2.57), P = 0.03]. It has been reported that polymorphisms in 5,10-methylenetetrahydrofolate reductase (MTHFR), 677C-->T (A222V), transcobalamin II (TCII), 776C-->G (P259R), are associated with pregnancy loss. Both variants were tested in this study. Neither showed evidence of significantly affecting the maternal risk of having a second trimester pregnancy loss. In conclusion, the MTHFD1 1958AA genotype may be an important maternal risk factor to consider during pregnancy.

Aborted Fetus↗
PubMed2005

Experimental RNomics in Aquifex aeolicus: identification of small non-coding RNAs and the putative 6S RNA homolog.

By an experimental RNomics approach, we have generated a cDNA library from small RNAs expressed from the genome of the hyperthermophilic bacterium Aquifex aeolicus. The library included RNAs that were antisense to mRNAs and tRNAs as well as RNAs encoded in intergenic regions. Substantial steady-state levels in A.aeolicus cells were confirmed for several of the cloned RNAs by northern blot analysis. The most abundant intergenic RNA of the library was identified as the 6S RNA homolog of A.aeolicus. Although shorter in size (150 nt) than its gamma-proteobacterial homologs (approximately 185 nt), it is predicted to have the most stable structure among known 6S RNAs. As in the gamma-proteobacteria, the A.aeolicus 6S RNA gene (ssrS) is located immediately upstream of the ygfA gene encoding a widely conserved 5-formyltetrahydrofolate cyclo-ligase. We identifed novel 6S RNA candidates within the gamma-proteobacteria but were unable to identify reasonable 6S RNA candidates in other bacterial branches, utilizing mfold analyses of the region immediately upstream of ygfA combined with 6S RNA blastn searches. By RACE experiments, we mapped the major transcription initiation site of A.aeolicus 6S RNA primary transcripts, located within the pheT gene preceding ygfA, as well as three processing sites.

Base Sequence↗