Growth Response of Lactobacillus rhamnosus Chloramphenicol-Resistant Strain ATCC 27773 to Pteroyl-Mono- and -Di-Glutamates.

This study investigated the effect of various concentrations of pteroyl-mono-γ-glutamate (PteGlu1) and pteroyl-di-γ-glutamate (PteGlu2) on the growth of Lactobacillus rhamnosus strains ATCC 7469 (wild-type strain) and ATCC 27773 (chloramphenicol-resistant strain) used for folate microbiological assays. At concentrations of 0.025-0.20 nmol/L, the growth of the chloramphenicol-resistant strain was stimulated to a greater extent by PteGlu1 than by PteGlu2, but the wild-type strain did not show such phenomena. L. rhamnosus ATCC 27773 bioassays were used to determine the total folate content of various foods treated with a chicken pancreas folate conjugase. This showed a significantly lower value when PteGlu1 was used as a calibrator than with PteGlu2. These results indicated that PteGlu2 should be the standard folate compound when chicken pancreas folate conjugase is used in preparing samples for L. rhamnosus ATCC 27773 bioassay.

Functional loss of the reduced folate carrier enhances the antitumor activities of novel antifolates with selective uptake by the proton-coupled folate transporter.

Uptake of 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolates with four or three bridge carbons [compound 1 (C1) and compound 2 (C2), respectively] into solid tumors by the proton-coupled folate transporter (PCFT) represents a novel therapeutic strategy that harnesses the acidic tumor microenvironment. Although these compounds are not substrates for the reduced folate carrier (RFC), the major facilitative folate transporter, RFC expression may alter drug efficacies by affecting cellular tetrahydrofolate (THF) cofactor pools that can compete for polyglutamylation and/or binding to intracellular enzyme targets. Human tumor cells including wild-type (WT) and R5 (RFC-null) HeLa cells express high levels of PCFT protein. C1 and C2 inhibited proliferation of R5 cells 3 to 4 times more potently than WT cells or R5 cells transfected with RFC. Transport of C1 and C2 was virtually identical between WT and R5 cells, establishing that differences in drug sensitivities between sublines were independent of PCFT transport. Steady-state intracellular [³H]THF cofactors derived from [³H]5-formyl-THF were depleted in R5 cells compared with those in WT cells, an effect exacerbated by C1 and C2. Whereas C1 and C2 polyglutamates accumulated to similar levels in WT and R5 cells, there were differences in polyglutamyl distributions in favor of the longest chain length forms. In severe combined immunodeficient mice, the antitumor efficacies of C1 and C2 were greater toward subcutaneous R5 tumors than toward WT tumors, confirming the collateral drug sensitivities observed in vitro. Thus, solid tumor-targeted antifolates with PCFT-selective cellular uptake should have enhanced activities toward tumors lacking RFC function, reflecting contraction of THF cofactor pools.

Comprehensive quantitative measurement of folate polyglutamates in human erythrocytes by ion pairing ultra-performance liquid chromatography/tandem mass spectrometry.

RATIONALE: The erythrocyte folate pool is reflective of an individual's long-term folate status; however, comprehensive quantitative determination of the various folate isoforms including polyglutamation (Glu(n)) status has posed an analytical problem. Factors complicating such analysis are the absence of authentic (isotope-labeled) standards and the large number of potential analytes. The present work presents high-throughput analytical methodology for the indirect comprehensive quantitation of the erythrocyte folate pool with commercially available standards. METHODS: The erythrocyte folate pool was determined comprehensively by utilizing a cascade of three complementary ultra-performance liquid chromatography (UPLC) tandem mass spectrometry (MS/MS) assays. In a first assay utilizing ion-pairing UPLC/MS/MS the relative (%) polyglutamation distribution (Glu(3-10)) of 5-methyltetrahydrofolate, tetrahydrofolate and 5-formyltetrahydrofolate is determined in a thermal extract obtained from packed erythrocytes, not requiring analytical standards. Quantitation of the erythrocyte folate pool was accomplished by performing two additional stable isotope dilution UPLC/MS/MS assays to determine whole blood and plasma folate content, utilizing commercially available [(13)C(5)]-labeled analogs of the Glu(1) analytes. Based on the values provided by each individual assay the comprehensive erythrocyte folate content could be calculated. RESULTS: The various assays have been validated for intra- and inter-run precision, accuracy, linearity and are robust. The method was sensitive enough to measure the comprehensive erythrocyte folate distribution in a Down's syndrome patient with extremely low folate, bearing the C677T mutation in the gene encoding for methylenetetrahydrofolate reductase. CONCLUSIONS: The erythrocyte folate pool can be comprehensively quantitated by running three complementary UPLC/MS/MS assays. The present assays are robust and allow for high-throughput analysis. The method can be utilized to support larger investigations that investigate the relationship between folate isoform and polyglutamation distribution and disease pathogenesis.

One-carbon metabolism in plants: characterization of a plastid serine hydroxymethyltransferase.

SHMT (serine hydroxymethyltransferase; EC 2.1.2.1) catalyses reversible hydroxymethyl group transfer from serine to H4PteGlun (tetrahydrofolate), yielding glycine and 5,10-methylenetetrahydrofolate. In plastids, SHMTs are thought to catalytically direct the hydroxymethyl moiety of serine into the metabolic network of H4PteGlun-bound one-carbon units. Genes encoding putative plastid SHMTs were found in the genomes of various plant species. SHMT activity was detected in chloroplasts in pea (Pisum sativum) and barley (Hordeum vulgare), suggesting that plastid SHMTs exist in all flowering plants. The Arabidopsis thaliana genome encodes one putative plastid SHMT (AtSHMT3). Its cDNA was cloned by reverse transcription-PCR and the encoded recombinant protein was produced in Escherichia coli. Evidence that AtSHMT3 is targeted to plastids was found by confocal microscopy of A. thaliana protoplasts transformed with proteins fused to enhanced green fluorescent protein. Characterization of recombinant AtSHMT3 revealed that substrate affinity for and the catalytic efficiency of H4PteGlu1-8 increase with n, and that H4PteGlu1-8 inhibit AtSHMT3. 5-Methyltetrahydrofolate and 5-formyltetrahydrofolate with one and five glutamate residues inhibited AtSHMT3-catalysed hydroxymethyl group transfer from serine to H4PteGlu6, with the pentaglutamylated inhibitors being more effective. Calculations revealed inhibition with 5-methyltetrahydrofolate or 5-formyltetrahydrofolate resulting in little reduction in AtSHMT3 activity under folate concentrations estimated for plastids.

Tomato gamma-glutamylhydrolases: expression, characterization, and evidence for heterodimer formation.

Folates typically have gamma-linked polyglutamyl tails that make them better enzyme substrates and worse transport substrates than the unglutamylated forms. The tail can be shortened or removed by the vacuolar enzyme gamma-glutamyl hydrolase (GGH). It is known that GGH is active only as a dimer and that plants can have several GGH genes whose homodimeric products differ functionally. However, it is not known whether GGH dimers dissociate under in vivo conditions, whether heterodimers form, or how heterodimerization impacts enzyme activity. These issues were explored using the GGH system of tomato (Solanum lycopersicum). Tomato has three GGH genes that, like those in other eudicots, apparently diverged recently. LeGGH1 and LeGGH2 are expressed in fruit and all other organs, whereas LeGGH3 is expressed mainly in flower buds. LeGGH1 and LeGGH2 homodimers differ in bond cleavage preference; the LeGGH3 homodimer is catalytically inactive. Homodimers did not dissociate in physiological conditions. When coexpressed in Escherichia coli, LeGGH1 and LeGGH2 formed heterodimers with an intermediate bond cleavage preference, whereas LeGGH3 formed heterodimers with LeGGH1 or LeGGH2 that had one-half the activity of the matching homodimer. E. coli cells expressing LeGGH2 showed approximately 85% reduction in folate polyglutamates, but cells expressing LeGGH3 did not, confirming that LeGGH2 can function in vivo and LeGGH3 cannot. The formation of LeGGH1-LeGGH2 heterodimers was demonstrated in planta using bimolecular fluorescence complementation. Plant GGH heterodimers thus appear to form wherever different GGH genes are expressed simultaneously and to have catalytic characteristics midway between those of the corresponding homodimers.

Concentration-dependent processivity of multiple glutamate ligations catalyzed by folylpoly-gamma-glutamate synthetase.

Folylpoly-gamma-glutamate synthetase (FPGS, EC 6.3.2.17) is an ATP-dependent ligase that catalyzes formation of poly-gamma-glutamate derivatives of reduced folates and antifolates such as methotrexate and 5,10-dideaza-5,6,7,8-tetrahydrofolate (DDAH 4PteGlu 1). While the chemical mechanism of the reaction catalyzed by FPGS is known, it is unknown whether single or multiple glutamate residues are added following each folate binding event. A very sensitive high-performance liquid chromatography method has been used to analyze the multiple ligation reactions onto radiolabeled DDAH 4PteGlu 1 catalyzed by FPGS to distinguish between distributive or processive mechanisms of catalysis. Reaction time courses, substrate trapping, and pulse-chase experiments were used to assess folate release during multiple glutamate additions. Together, the results of these experiments indicate that hFPGS can catalyze the processive addition of approximately four glutamate residues to DDAH 4PteGlu 1. The degree of processivity was determined to be dependent on the concentration of the folate substrate, thus suggesting a mechanism for the regulation of folate polyglutamate synthesis in cells.

Nutrients involved in one-carbon metabolism and risk of breast cancer among premenopausal women.

Folate, vitamin B6, vitamin B12, methionine, choline, and betaine are nutrients involved in one-carbon metabolism and have been hypothesized to reduce the risk of breast cancer. However, previous epidemiologic studies on most of these nutrients and breast cancer risk have been inconclusive and have included primarily postmenopausal women. No study has examined choline and betaine in relation to breast cancer risk. Therefore, we examined the intake of these nutrients in relation to breast cancer risk among 90,663 premenopausal women ages 26 to 46 years in 1991 in the Nurses' Health Study II. Nutrient intake was assessed with a validated food frequency questionnaire in 1991, 1995, and 1999. During 12 years of follow-up from 1991 to 2003, we documented 1,032 incident cases of invasive breast cancer. Overall, none of the nutrients was associated with risk of breast cancer. The results were similar by levels of alcohol intake and folate intake and for estrogen receptor-negative breast cancer. In conclusion, we found no evidence that higher intakes of nutrients involved in one-carbon metabolism reduce risk of breast cancer among premenopausal women.

Comparative genomics of bacterial and plant folate synthesis and salvage: predictions and validations.

BACKGROUND: Folate synthesis and salvage pathways are relatively well known from classical biochemistry and genetics but they have not been subjected to comparative genomic analysis. The availability of genome sequences from hundreds of diverse bacteria, and from Arabidopsis thaliana, enabled such an analysis using the SEED database and its tools. This study reports the results of the analysis and integrates them with new and existing experimental data. RESULTS: Based on sequence similarity and the clustering, fusion, and phylogenetic distribution of genes, several functional predictions emerged from this analysis. For bacteria, these included the existence of novel GTP cyclohydrolase I and folylpolyglutamate synthase gene families, and of a trifunctional p-aminobenzoate synthesis gene. For plants and bacteria, the predictions comprised the identities of a 'missing' folate synthesis gene (folQ) and of a folate transporter, and the absence from plants of a folate salvage enzyme. Genetic and biochemical tests bore out these predictions. CONCLUSION: For bacteria, these results demonstrate that much can be learnt from comparative genomics, even for well-explored primary metabolic pathways. For plants, the findings particularly illustrate the potential for rapid functional assignment of unknown genes that have prokaryotic homologs, by analyzing which genes are associated with the latter. More generally, our data indicate how combined genomic analysis of both plants and prokaryotes can be more powerful than isolated examination of either group alone.

Impaired spatial memory in APP-overexpressing mice on a homocysteinemia-inducing diet.

Consumption of a diet that significantly elevates homocysteine (homocysteinemia) induces cell death in the CA3 hippocampal subfield in amyloid precursor protein (APP) over-expressing transgenic mice but not in wild-type controls. We assessed behavioral and other neuropathological effects of a homocysteinemia-inducing diet in aged APP-overexpressing mice. Starting at 16-18 months of age, mice were fed either a treatment diet lacking folate, choline, and methionine, and supplemented with homocysteine, or a control diet containing normal amounts of folate, choline and methionine but no homocysteine. After 5 months on the experimental diets, performance on a delayed non-matching-to-position working-memory task was unimpaired. In contrast, spatial reference memory in the water maze was impaired in transgenic mice on the treatment diet. Transgenic mice had higher homocysteine levels than wild-type mice even when fed the control diet, suggesting an effect of genotype on homocysteine metabolism. Methyl-donor deficiency did not alter amyloid deposition in the transgenic mice. These results suggest that disrupted homocysteine metabolism may induce Abeta-associated memory impairments and neurodegeneration in APP overexpressing mice.

Folate absorption from folate-fortified and processed foods using a human ileostomy model.

Data on folate absorption from food from validated human studies using physiological folate doses are still needed to estimate dietary requirements and to formulate recommendations. The aim of the present work was to study the effects from fortified and processed foods on folate absorption in ileostomy volunteers (n 9) using the area under the plasma concentration curve (AUC) and kinetic modelling. Using a standardized single-dose protocol, dairy products fortified with a candidate fortificant (6S)-5-methyltetrahydrofolate ((6S)-5-CH3-H4folate), folic acid-fortified bread and a dessert crème containing natural yeast folate polyglutamates were compared with folate supplements. Absorbed folate was estimated by AUC and a kinetic model, and non-absorbed folate by ileostomal folate excretion. Median apparent absorption from test foods ranged from 55 to 86 %. Added folate-binding proteins (FBP) significantly reduced folate absorption from dairy products, as in the absence of FBP, AUC-dose-corrected ratios were increased and ileal folate excretion decreased. After in vivo gastrointestinal passage of dairy products containing FBP, up to 43 % of the ingested FBP was found in ileostomal effluent. Folate absorption was similar for (6S)-5-CH3-H4folate fortificant from fermented milk and for folic acid from fortified bread. Folic acid, ingested as food fortificant in bread, was significantly less absorbed compared with an isolated supplement. We conclude that all tested foods were suitable matrices for folate fortification. However, dairy products, fortified with the new candidate fortificant (6S)-5-CH3-H4folate, are suitable if no active FBP is present.

Properties of human and rabbit cytosolic serine hydroxymethyltransferase are changed by single nucleotide polymorphic mutations.

Serine hydroxymethyltransferase (SHMT) is a key enzyme in the formation and regulation of the folate one-carbon pool. Recent studies on human subjects have shown the existence of two single nucleotide polymorphisms that may be associated with several disease states. One of these mutations results in Ser394 being converted to an Asn (S394N) and the other in the change of Leu474 to a Phe (L474F). These mutations were introduced into the cDNA for both human and rabbit cytosolic SHMT and the mutant enzymes expressed and purified from an Escherichia coli expression system. The mutant enzymes show normal values for kcat and Km for serine. However, the S394N mutant enzyme has increased dissociation constant values for both glycine and tetrahydrofolate (tetrahydropteroylglutamate) and its pentaglutamate form compared to wild-type enzyme. The L474F mutant shows lowered affinity (increased dissociation constant) for only the pentaglutamate form of the folate ligand. Both mutations result in decreased rates of pyridoxal phosphate addition to the mutant apo enzymes to form the active holo enzymes. Neither mutation significantly affects the stability of SHMT or the rate at which it converts 5,10-methenyl tetrahydropteroyl pentaglutamate to 5-formyl tetrahydropteroyl pentaglutamate. Analysis of the structures of rabbit and human SHMT show how mutations at these two sites can result in the observed functional differences.

Controlled modulation of folate polyglutamyl tail length by metabolic engineering of Lactococcus lactis.

The dairy starter bacterium Lactococcus lactis is able to synthesize folate and accumulates >90% of the produced folate intracellularly, predominantly in the polyglutamyl form. Approximately 10% of the produced folate is released into the environment. Overexpression of folC in L. lactis led to an increase in the length of the polyglutamyl tail from the predominant 4, 5, and 6 glutamate residues in wild-type cells to a maximum of 12 glutamate residues in the folate synthetase overproducer and resulted in a complete retention of folate in the cells. Overexpression of folKE, encoding the bifunctional protein 2-amino-4-hydroxy-6-hydroxymethyldihydropteridine pyrophosphokinase and GTP-cyclohydrolase I, resulted in reduction of the average polyglutamyl tail length, leading to enhanced excretion of folate. By simultaneous overexpression of folKE and folC, encoding the enzyme folate synthetase or polyglutamyl folate synthetase, the average polyglutamyl tail length was increased, again resulting in normal wild-type distribution of folate. The production of bioavailable monoglutamyl folate and almost complete release of folate from the bacterium was achieved by expressing the gene for gamma-glutamyl hydrolase from human or rat origin. These engineering studies clearly establish the role of the polyglutamyl tail length in intracellular retention of the folate produced. Also, the potential application of engineered food microbes producing folates with different tail lengths is discussed.

Single oral doses of 13C forms of pteroylmonoglutamic acid and 5-formyltetrahydrofolic acid elicit differences in short-term kinetics of labelled and unlabelled folates in plasma: potential problems in interpretation of folate bioavailability studies.

Single (13)C6-labelled doses of pteroylmonoglutamic acid (PteGlu; 634 nmol) or 5-formyltetrahydrofolic acid (431-569 nmol) were given to fasted adult volunteers, and the rise in total and (13)C-labelled plasma 5-methyltetrahydrofolic acid metabolite monitored over 8 h by HPLC and liquid chromatography-MS. The dose-adjusted area under the curve (AUC) for total (labelled plus unlabelled) plasma 5-methyltetrahydrofolic acid following a 5-formyltetrahydrofolic acid test dose was 155 % that obtained following a PteGlu test dose. Surprisingly, an average 60 and 40 % of the total plasma 5-methyltetrahydrofolic acid response to [(13)C6]PteGlu and [(13)C6]5-formyltetrahydrofolic acid, respectively, was unlabelled; an observation never before reported. Short-term kinetics of plasma [(13)C6]5-methyltetrahydrofolic acid showed a slower initial rate of increase in plasma concentration and longer time to peak following an oral dose of [(13)C6]PteGlu compared with that for an oral dose of [(13)C6]5-formyltetrahydrofolic acid, while the [(13)C6]5-methyltetrahydrofolic acid AUC for [(13)C6]5-formyltetrahydrofolic acid was 221 % that for [(13)C6]PteGlu. These data indicate that PteGlu and 5-formyltetrahydrofolic acid, which are thought to be well absorbed (about 90 %) at physiological doses, exhibit dramatically different rates and patterns of plasma response. A limitation in the rate of reduction of PteGlu before methylation could result in slower mucosal transfer of [(13)C6]5-methyltetrahydrofolic acid derived from [(13)C6]PteGlu into the plasma. This, when coupled with an observed similar plasma clearance rate for [(13)C6]5-methyltetrahydrofolic acid metabolite derived from either folate test dose, would yield a comparatively smaller AUC. These findings suggest potential problems in interpretation of absorption studies using unlabelled or labelled folates where the rate of increase, the maximum increase, or the AUC, of plasma folate is employed for test foods (mainly reduced folates) v. a 'reference dose' of PteGlu.

Methotrexate polyglutamation may lack prognostic significance in children with B-cell precursor acute lymphoblastic leukemia treated with intensive oral methotrexate.

BACKGROUND: The purpose of this study was to determine if a correlation exists between clinical outcome and accumulation and polyglutamation of methotrexate by lymphoblasts in vitro in children with B-cell precursor acute lymphoblastic leukemia (BCP-ALL). PATIENTS AND METHODS: The amount of accumulated methotrexate and of long-chain methotrexate polyglutamates (MTXPG(3-7)) by lymphoblasts was determined in 52 children newly diagnosed with BCP-ALL after incubation with 1 micromol/L [(3)H]MTX for 24 hours in vitro. All patients then received intensive multiagent chemotherapy that used divided-dose oral methotrexate during consolidation and intensive continuation and standard oral weekly methotrexate during maintenance. RESULTS: Eight patients had a bone marrow relapse at a median of 40.4 months (range 18.5-48.3 months) after diagnosis. The median follow-up for the remaining 44 patients is 69.0 months (range 22-92.8 months). There was no significant difference in the amount of accumulated methotrexate (1450.0 +/- 896.3 vs. 640 +/- 472.5 pmol/10 cells) or of accumulated MTXPG (1450.0 +/- 919.4 vs. 617.4 +/- 482.7 pmol/10(9) cells) (median +/- semi-interquartile ranges) between patients who relapsed and those who remained in continuous complete remission. The estimated 5-year event-free survival rate for patients whose lymphoblasts accumulated more than 500 pmol MTXPG(3-7)/10(9) cells was 80.0% +/- 7.3% versus 90.5% +/- 6.4% for those whose lymphoblasts accumulated less than 500 pmol MTXPG(3-7)/10(9) cells. CONCLUSIONS: In the context of effective prolonged divided-dose oral methotrexate-based therapy in the treatment of BCP-ALL, methotrexate accumulation and polyglutamation no longer seem to have prognostic significance.

Molecular, biochemical, and cellular pharmacology of pemetrexed.

Pemetrexed is a new-generation antifolate that in its higher polyglutamyl forms is a potent, direct inhibitor of thymidylate synthase and, to a lesser extent, glycinamide ribonucleotide transformylase. Activity of the drug may be partially preserved under conditions in which cells are highly resistant to other thymidylate synthase inhibitors, possibly because of premetrexed's secondary inhibitory effects on purine synthesis. Pemetrexed inhibition of dihydrofolate reductase is not of pharmacologic importance. Pemetrexed has high affinity for the reduced folate carrier and folate receptor and is among the most potent substrates for folylpolyglutamate synthetase. These properties result in rapid accumulation of the free drug in cells with the rapid formation of high levels of the active polyglutamyl congeners. Pemetrexed activity is modulated by natural folates within cells that compete for polyglutamation at the level of folylpolyglutamate synthetase. Cells resistant to methotrexate because of impaired transport via the reduced folate carrier may retain partial sensitivity to pemetrexed. This is due to concurrent diminished transport of physiologic reduced folates and contraction of the cellular folate pool, thereby relaxing the usual level of suppression of pemetrexed polyglutamation. The risk of pemetrexed toxicity is increased when cellular folates are suboptimal. This is best monitored by assessment of blood homocysteine levels, and can be diminished by the coadministration of folic acid.

Ligand binding characteristics of two molecular forms, one equipped with a hydrophobic glycosyl phosphatidylinositol tail, of the folate binding protein purified from human milk.

Two molecular forms of the folate binding protein were isolated and purified from human milk by a combination of cation exchange- and affinity chromatography. One protein (27 kDa) was a cleavage product of the other 100 kDa protein as evidenced by N-terminal amino acid sequence homology and a reduction in the molecular size of the latter protein to 27 kDa after cleavage of its hydrophobic glycosylphosphatidylinositol tail by phosphatidylinositol-specific phospholipase C. High-affinity binding of [3H]folate was characterized by upward convex Scatchard plots and increasing ligand binding affinity with decreasing concentrations of both proteins. Downward convex Scatchard plots and binding affinities showing no dependence on the protein concentration were, however, observed in highly diluted solutions of both proteins. Radioligand binding was inhibited by folate analogs, and dissociation of radioligand was slow at pH 7.4 but rapid and complete at pH 5.0 and 3.5. Ligand binding quenched the tryptophan fluorescence of the 27 kDa protein suggesting that tryptophan is present at the binding site and/or ligand binding induces a conformation change that affects tryptophan environment in the protein. The 27 kDa protein representing soluble folate binding protein exhibited a greater affinity for ligand binding than the 100 kDa protein which possesses a hydrophobic tail identical to the one that anchors the folate receptor to the cell membrane.

An examination of polymorphic genes and folate metabolism in mothers affected by a spina bifida pregnancy.

The effect of four polymorphic genes of folate-dependent methionine biosynthesis have been investigated in mothers affected by a neural tube defect pregnancy (NTD) and matched controls. The influence of the various genotypes on total red cell 5-methyl-H(4)folate,5,10-methenyl-H(4)folate, and 5-formyl-H(4)folate is reported, as is the effect on homocysteine and radioassay folate in both serum and red cells. All of the single nucleotide polymorphisms studied would seem to contribute to the cellular folate profile in some way. From the data presented, and from the work of others, it is likely that C677T 5,10-methylenetetrahydrofolate reductase is the most important of these polymorphisms. Control mother folate profiles seem reasonably predictive of any given methionine cycle mutation, but profiles in NTD mothers do not. On this basis, it seems likely that some other, as yet unidentified folate lesion is causal for NTD. In NTD-C677T 5,10-methylenetetrahydrofolate reductase in particular, indexes of folate depletion such as high-performance liquid chromatography (HPLC) folate level, oligo-gamma-glutamyl chain length, homocysteine, and radioassay folate values all seem to deteriorate with increased mutant allele carriage. This indicates that this folate polymorphism may provide a critical threshold effect that helps to promote NTD occurrence in the presence of another, as yet unidentified folate-related factor. In more general terms, on a by genotype basis, all 11 genotypes studied give NTD mothers a higher homocysteine compared to controls. Furthermore, a trend that is less universal indicates that NTD mothers have higher 5,10-methenyl-H(4)folate and 5-methyl-H(4)folate levels and lower 5-formyl-H(4)folate and H(4)PteGlu(1) levels than do controls. One of the most consistent, and possibly specific, differences between participant groups is a statistically significant elevation of 5,10-methenyl-H(4)folate in NTD mothers (affects three genotypes). Possible interpretations of this finding are discussed.

Antifolate resistance and its circumvention by new analogues.

We have established human leukemia cell lines made resistant to various antifolate drugs and analyzed resistance mechanisms developed in these cells at the cellular and molecular levels. The cells acquired resistance to antifolate drug(s) through: (1) impaired drug uptake via the reduced folate carrier, (2) increased activity of the target enzymes[dihydrofolate reductase(DHFR) or thymidylate synthase(TS)] resulted from a concomitant amplification and overexpression of their gene, (3) induction of a variant DHFR with a low affinity for antifolate drug(s) used for the selection of resistance, and (4) defective polyglutamation. Each resistance mechanism was not necessarily induced at random, but appeared to relate to the biochemical and pharmacological properties of the drug exposed, biological dispositions of the cells, drug-exposure manners to, or culture conditions of the cells. Since it has been shown that a minor modification at the specified position of the folate structure resulted in a drastic change in its pharmacological properties, many new compounds have been rationally designed on the basis of the knowledge of relationships between structure modifications and pharmacological properties. The step-by-step approach to the development of new analogues led to the discoveries of several promising antifolate drugs such as trimetrexate and raltitrexed, which can overcome the acquired and natural resistance to methotrexate, a classical antifolate, and clinical trials of these newer classes of antifolate compounds are currently underway.

Polyglutamation of antifolates is not required for induction of extracellular release of adenosine or expression of their anti-inflammatory effects.

Methotrexate (MTX) exerts an anti-inflammatory effect, reportedly by enhancing the release of adenosine, through an accumulation of 5-amino-4-imidazolecarboxamide ribonucleotide (AICAR). To examine the role of polyglutamation in promoting anti-inflammatory effects by antifolates, we tested whether a new antifolate designed to be resistant to intracellular polyglutamation (MX-68) exhibited anti-inflammatory effects and stimulated adenosine release. Both MX-68 and MTX (at concentrations greater than 0.1 microM) increased the release of adenosine from Daudi cells in vitro. Inhibition of AICAR synthesis suppressed adenosine release by MX-68 and MTX. The anti-inflammatory effects of antifolates were estimated using the murine air pouch model, in which a BALB/c mouse was intraperitoneally injected with MX-68 or MTX once a week for 3 weeks. MX-68 (0.5 mg kg(-1) week(-1)), as well as MTX, inhibited infiltration of leukocytes into the air pouch. This inhibitory effect was suppressed in the presence of an adenosine A(2) receptor antagonist, 3,7-dimethyl-1-propargylxanthine (DMPX). These results suggest that MX-68, like MTX, exerts anti-inflammatory effects through the accumulation of AICAR and release of adenosine. These results suggest that polyglutamation of antifolate is not required for expression of anti-inflammatory effects.

Altered folate metabolism and disposition in mothers affected by a spina bifida pregnancy: influence of 677c --> t methylenetetrahydrofolate reductase and 2756a --> g methionine synthase genotypes.

Periconceptional folate prevents spina bifida although the mechanisms involved are unclear. We present the genotype frequency for the 677 ct methylenetetrahydrofolate reductase (MTHFR) and 2756ag methionine synthase (MetSyn) polymorphisms. Calculated odds ratios (OR) show that neither the homozygous recessive genotype, carriage of the mutant allele, nor frequency of the mutant allele represent significantly increased risk for neural tube defect (NTD). This is true for both polymorphisms. Simultaneous carriage of t and g alleles is also not a significantly increased risk for NTD. OR and 95% CI for carriage of (i) t allele, (ii) g allele, and (iii) simultaneous carriage of t and g alleles in NTD are 0.89 (0.28-2.82), 0.97 (0.28-3.30), and 0.61 (0.11-3.52), respectively. OR and 95% CI for frequency of t and g alleles are 0.94 (0.42-2.13) and 0.88 (0. 29-2.67), respectively. Unlike some previous studies, we could not detect a significantly increased risk for NTD conferred by the 677ct MTHFR tt genotype; OR 0.98 (0.19-6.49). Differences were found to exist in the circulating whole blood folate profile: total formyl-H(4)PteGlu was significantly higher than total 5-methyl-H(4)PteGlu in control (P = 0.036) but not NTD blood. When broken down into the various 677 ct MTHFR and 2756ag MetSyn genotypes, carriage of the 677ct MTHFR allele appears to affect formyl-H(4)PteGlu metabolism in non-NTD mothers. In addition, NTD mothers exhibited noticeably lower formyl-H(4)PteGlu levels compared to controls; these effects, however, were not significant. 2756ag MetSyn is similarly associated with an altered formyl-H(4)PteGlu disposition. The ag genotype had significantly more formyl-H(4)PteGlu relative to 5-methyl-H(4)PteGlu than wildtype 2756ag MetSyn (P = 0.024). This heterozygous increase in the relative formyl-H(4)PteGlu level holds true for controls only; no such relationship occurred in NTD samples. Folyl hexaglutamates are the active cellular coenzyme forms. We showed that where 5-methyl-H(4)PteGlu(6) predominates, Hcy levels are highest. As the relative abundance of formyl-H(4)PteGlu(6) increased, so Hcy decreased, presumably due to increased Hcy remethylation, a process in which 5-methyl-H(4)PteGlu(6) is demethylated and downstream folates like formyl-H(4)PteGlu(6) are produced. The negative linear association between the hexaglutamate ratio (formyl-H(4)PteGlu(6)/5-methyl-H(4)PteGlu(6)) and Hcy is significant for control (r = -0.64, P = 0.003) but not NTD samples. This effect, centering on Hcy remethylation, is supported by a statistically elevated formyl-H(4)PteGlu(6) to 5-methyl-H(4)PteGlu(6) level in controls relative to NTDs (P = 0.047). The overall (polymorphism independent) effect of exogenous 5,10-methenyl-H(4)PteGlu(1) substrate on the cellular folate profile was to preferentially increase formyl-H(4)PteGlu, while exogenous 5-methyl-H(4)PteGlu(1) substrate dramatically increased metabolic production of 5, 10-methylene-H(4)PteGlu. The following differences were observed between NTD and control samples: (i) a reduced expansion of the formyl-H(4)PteGlu(6) pool in NTD with exogenous 5, 10-methenyl-H(4)PteGlu(1) (P = 0.0005 for control expansion, NS for NTD increase); (ii) a reduced initial expansion of the 5, 10-methylene-H(4)PteGlu pool in NTD following treatment with exogenous 5-methyl-H(4)PteGlu(1) substrate (difference between subject groups; P = 0.031). In addition, taking polymorphisms into account, lysate from NTD-MTHFR wildtypes utilized less exogenous 5-methyl-H(4)PteGlu(1) substrate than control-MTHFR wildtypes in the short (P = 0.011) and long term (P = 0.036). Commensurate with this latter effect, the initial production of 5,10-methylene-H(4)PteGlu due to exogenous 5-methyl-H(4)PteGlu(1) substrate was significantly reduced in the NTD-MTHFR wildtype (P = 0.037). These two MTHFR wildtype effects imply that the 677 ct polymorphism is not the only mutation affecting folate metabolism in NTD mothers. (ABSTRACT TRUNCATED)