Folate Polyglutamates Research Articles

Folylpolyglutamate synthetase inactivation in relapsed ALL induces a druggable folate metabolic vulnerability

AimsThe antifolate methotrexate (MTX) is an anchor drug used in acute lymphoblastic leukemia (ALL) with poorly understood chemoresistance mechanisms in relapse. Herein we find decreased folate polyglutamylation network activities and inactivating FPGS mutations, both of which could induce MTX resistance and folate metabolic vulnerability in relapsed ALL. MethodsWe utilized integrated systems biology analysis of transcriptomic and genomic data from relapse ALL cohorts to infer hidden ALL relapse drivers and related genetic alternations during clonal evolution. The drug sensitivity assay was used to determine the impact of relapse-specific FPGS mutations on sensitivity to different antifolates and chemotherapeutics in ALL cells. We used liquid chromatography-mass spectrometry (LC-MS) to quantify MTX and folate polyglutamate levels in folylpoly-γ-glutamate synthetase (FPGS) mutant ALL cells. Enzymatic activity and protein degradation assays were also conducted to characterize the catalytic properties and protein stabilities of FPGS mutants. An ALL cell line-derived mouse leukemia xenograft model was used to evaluate the in vivo impact of FPGS inactivation on leukemogenesis and sensitivity to the polyglutamatable antifolate MTX as well as non-polyglutamatble lipophilic antifolate trimetrexate (TMQ). ResultsWe found a significant decrease in folate polyglutamylation network activities during ALL relapse using RNA-seq data. Supported by functional evidence, we identified multifactorial mechanisms of FPGS inactivation in relapsed ALL, including its decreased network activity and gene expression, focal gene deletion, impaired catalytic activity, and increased protein degradation. These deleterious FPGS alterations induce MTX resistance and inevitably cause marked intracellular folate shrinkage, which could be efficiently targeted by a polyglutamylation-independent lipophilic antifolate TMQ in vitro and in vivo. ConclusionsMTX resistance in relapsed ALL relies on FPGS inactivation, which inevitably induces a folate metabolic vulnerability, allowing for an efficacious antifolate ALL treatment strategy that is based upon TMQ, thereby surmounting chemoresistance in relapsed ALL.

Cellular pharmacokinetic of methotrexate and its modulation by folylpolyglutamate synthetase and γ-glutamyl hydrolase in tumor cells.

Clinical studies showed that prolonged infusion of methotrexate (MTX) leads to more severe adverse reactions than short infusion of MTX at the same dose. We hypothesized that it is the saturation of folate polyglutamate synthetase (FPGS) at high MTX concentration that limits the intracellular synthesis rate of methotrexate polyglutamate (MTX-PG). Due to a similar accumulation rate, a longer infusion duration may increase the concentration of MTX-PG and, result in more serious adverse reactions. In this study, we validated this hypothesis. A549, BEL-7402 and MHCC97H cell lines were treated with MTX at gradient concentrations. Liquid chromatograph-mass spectrometer (UPLC-MS/MS) was used to quantify the intracellular concentration of MTX-PG and the abundance of FPGS and γ-glutamyl hydrolase (GGH). High quality data were used to fit the cell pharmacokinetic model. Both cell growth inhibition rate and intracellular MTX-PG concentration showed a nonlinear relationship with MTX concentration. The parameter Vmax in the model, which represents the synthesis rate of MTX-PG, showed a strong correlation with the abundance of intracellular FPGS. According to the model fitting results, it was confirmed that the abundance of FPGS is a decisive factor limiting the synthesis rate of MTX-PG. The proposed hypothesis was verified in this study. In addition, based on the intracellular metabolism, a reasonable explanation was provided for the correlation between the severity of adverse reactions of MTX and infusion time. This study provides a new strategy for the individualized treatment and prediction of efficacy/side effects of MTX.

Addition of exogenous γ-glutamyl hydrolase eliminates the need for lengthy incubation of whole blood lysate for quantitation of folate vitamers by high performance liquid chromatography-tandem mass spectrometry

BackgroundMeasurement of folate monoglutamates by HPLC-MS/MS in whole blood lysate (WBL) requires lengthy incubation prior to analysis, risking degradation of labile folate vitamers. ObjectiveWe explored whether addition of a commercially available recombinant exogenous γ-glutamyl hydrolase (exoGGH) enzyme reduced the required incubation time of WBL for measurement of folate as monoglutamates. MethodsFor conventional deglutamylation of polyglutamates, WBL was incubated 4 h at 37°C. Alternatively, we added exoGGH to WBL at varying concentrations (1–10 µg/mL) and incubation times (0–90 min). We also investigated modifications to the sample diluent (pH, ascorbic acid vs. sodium ascorbate, and ascorbate concentration). Finally, we tested the effect of the enzyme in different sample types: WBL from frozen whole blood vs. frozen WBL or vs. frozen washed red blood cells (RBC). Samples (n ≤ 15 per experiment) were analyzed by HPLC-MS/MS for 6 folate monoglutamates and 5-methyltetrahydrofolate diglutamate. ResultsOptimal deconjugation of folate polyglutamates was achieved using 1% ascorbic acid and 5 µg enzyme/mL WBL, requiring ≤ 30 min incubation time to achieve complete folate recovery as monoglutamates. This treatment resulted in similar folate concentrations as conventional deglutamylation (4 h at 37°C). The exoGGH enzyme was effective in samples stored frozen as whole blood and as WBL. However, extended thaw time of whole blood resulted in 5-methyltetrahydrofolate loss and unacceptable changes to non-methyl folate concentration. Total folate (with exoGGH) measured in washed RBC was ∼15% lower than RBC folate calculated from WBL concentrations (conventional deglutamylation). ConclusionsUse of exoGGH minimized incubation time and thus may avoid degradative losses of labile folate forms during sample preparation. The lower folate results in washed RBC may be due to inadequate packing of RBC among other unidentified factors. A larger study is required to confirm lack of differences in folate concentrations determined with and without the use of exoGGH.

Folate Forms in RBC and Whole-Blood Lysates Appear Stable When Stored Frozen for 2 Years

BackgroundThe use of RBC lysate (RBC-Lys) eliminates the need for serum folate and hematocrit (Hct) measurement to calculate RBC folate. Information on the long-term frozen storage stability of RBC-Lys is missing. ObjectivesWe aimed to assess the comparability of RBC folate forms in whole-blood lysate (WB-Lys) and RBC-Lys and the folate stability in both matrices. MethodsWe prepared conventional WB-Lys (1:11 dilution with 1% ascorbic acid) and RBC-Lys (1:11 dilution of washed and saline-diluted RBCs with 1% ascorbic acid) from EDTA blood (n = 60 adult donors) and stored lysates at −70°C until analysis at baseline (1 wk), 3, 6, 12, and 24 mo. Before analysis by HPLC–tandem MS, we incubated the WB-Lys (4 h at 37°C) and treated the RBC-Lys with human recombinant γ-glutamyl hydrolase for folate polyglutamate deconjugation. We analyzed RBC-Lys samples for hemoglobin (Hb) (same aliquot) to normalize for the preanalytical dilution; Hb-folate was converted to RBC folate for each folate form using the mean corpuscular Hb concentration. We analyzed Hct as well as folate forms in matching serum samples for traditional RBC folate calculation. We conducted descriptive data analyses (correlation, Bland–Altman plot, Deming regression). ResultsAt baseline, results for RBC folate forms derived from WB-Lys compared with RBC-Lys samples showed excellent correlation (Pearson r ≥ 0.97). Mean ± SD concentrations compared well for total folate (WB-Lys: 886 ± 255 compared with RBC-Lys: 899 ± 271 nmol/L), 5-methyltetrahydrofolate (WB-Lys: 831 ± 258 compared with RBC-Lys: 843 ± 276 nmol/L), and nonmethyl folate (WB-Lys: 53.3 ± 74.4 compared with RBC-Lys: 52.9 ± 70.7 nmol/L), but were 17% higher in RBC-Lys for pyrazino-s-triazine derivative of 4α-hydroxy-5-CH3-H4folate (MeFox) (WB-Lys: 147 ± 44.1 compared with RBC-Lys: 172 ± 53.5 nmol/L). Frozen storage of WB-Lys and RBC-Lys samples for ≤24 mo showed ≤5%, ≤5%, ≤13%, and ≤11% change in total folate, 5-methyltetrahydrofolate, nonmethyl folate, and MeFox, respectively. ConclusionsErythrocyte folate forms appear to be stable in RBC-Lys samples stored frozen at −70°C for ≤2 y. The relatively small changes in folate concentrations over time were comparable between RBC-Lys and conventionally prepared WB-Lys samples.

Folate Forms in Red Blood Cell Lysates and in Intact Washed Red Blood Cells Are Stable for a Few Days During Refrigerated Storage

Abstract Objectives We investigated whether folate forms are stable in washed red blood cells (RBC) stored refrigerated for up to 9 days relative to RBC-lysates in ascorbic acid under the same storage conditions. Methods We prepared washed RBCs from freshly collected EDTA blood (n = 6 donors). For RBC-lysate 1, we diluted RBCs with saline (1/2 dilution), mixed well and prepared lysates (1/11 dilution) with 1% ascorbic acid (2 vials/time point; n = 12 vials/donor) for storage at 4°C for ≤ 9 days (baseline, 1, 2, 3, 6, and 9). For RBC-lysate 2, we aliquoted ∼0.5 mL washed RBCs (1 vial/time point; n = 5 vials/donor) for storage at 4°C for ≤9 days (1, 2, 3, 6, and 9). When refrigerated storage of samples was completed, RBC lysate 1 samples were frozen at −70°C, while the washed RBCs were diluted with saline (1/2 dilution), mixed well and RBC-lysates 2 were prepared (1/11 dilution) with 1% ascorbic acid (2 vials/time point; n = 10 vials/donor). All samples were stored at −70°C until analysis. At the time of analysis, we processed samples (2 replicates/time point; n = 2 days) for folate polyglutamate deconjugation using recombinant exo γ-glutamyl hydrolase, and conducted sample clean-up by automated solid phase extraction prior to analysis by LC-MS/MS. Results We found negligible losses of major folate forms after overnight refrigerated storage, and folate losses gradually increased over time (∼5% by day 6). The loss of 5-methyltetrahydrofolate and total folate (mean ± SD) after 2 days of storage for RBC lysate 1 was 1.2% ± 1.5% and 1.0% ± 1.3%, and for RBC-lysate 2 was 2.2% ± 1.1% and 2.7% ± 1.9%, respectively. The baseline concentration of non-methyl folate (sum of minor folate forms: 5-formyltetrahydrofolate [<LOD], tetrahydrofolate, and 5, 10-methenyltetrahydrofolate) was small (<3.0 nmol/L) in both sample types, and we noticed a slight increase (∼10%) after overnight storage. 5,10-Methenyltetrahydrofolate appeared to gradually convert to tetrahydrofolate upon storage in RBC-lysate 2 samples. We observed no noticeable changes for MeFox in either sample type. Conclusions Overnight refrigerated storage of RBC-lysates or washed RBCs is feasible and avoids notable losses of folate forms. Prolonged refrigerated storage promotes interconversions of minor folate forms. Funding Sources This work was supported by direct appropriations from U.S. Congress.

Genetically determined folate deficiency is associated with abnormal hepatic folate profiles in the spontaneously hypertensive rat.

Increased levels of plasma cysteine are associated with obesity and metabolic disturbances. Our recent genetic analyses in spontaneously hypertensive rats (SHR) revealed a mutated Folr1 (folate receptor 1) as the quantitative trait gene associated with diminished renal Folr1 expression, lower plasma folate levels, hypercysteinemia, hyperhomocysteinemia and metabolic disturbances. To further analyse the effects of the Folr1 gene expression on folate metabolism, we used mass spectrometry to quantify folate profiles in the plasma and liver of an SHR-1 congenic strain, with wild type Folr1 allele on the SHR genetic background, and compared them with the SHR strain. In the plasma, concentration of 5-methyltetrahydrofolate (5mTHF) was significantly higher in SHR-1 congenic rats compared to SHR (60+/-6 vs. 42+/-2 nmol/l, P<0.01) and 5mTHF monoglutamate was the predominant form in both strains (>99 % of total folate). In the liver, SHR-1 congenic rats showed a significantly increased level of 5mTHF and decreased concentrations of dihydrofolate (DHF), tetrahydrofolate (THF) and formyl-THF when compared to the SHR strain. We also analysed the extent of folate glutamylation in the liver. Compared with the SHR strain, congenic wild-type Folr1 rats had significantly higher levels of 5mTHF monoglutamate. On the other hand, 5mTHF penta- and hexaglutamates were significantly higher in SHR when compared to SHR-1 rats. This inverse relationship of rat hepatic folate polyglutamate chain length and folate sufficiency was also true for other folate species. These results strongly indicate that the whole body homeostasis of folates is substantially impaired in SHR rats compared to the SHR-1 congenic strain and might be contributing to the associated metabolic disturbances observed in our previous studies.

Continuous infusion fluorouracil (5FU) in heavily pre-treated mCRPC patients.

257 Background: Pts with mCRPC resistant to novel hormones (abi/enza), and taxane chemotherapy are challenging. Extensive pre-treatment typically poses an issue for clinical trials, and there is no consensus regarding how to manage patients who no longer respond to conventional therapies but maintain a good performance status. Methods: 13 mCRPC pts were treated with 5FU at 200mg/m2 continuous infusion as a novel treatment option for those with heavily pretreated mCRPC. Data was retrospectively collected at Tulane Cancer Center for patient treatment history, and laboratory parameters. Results: All treated patients had prior exposure to an average of 8.7 lines of treatment. 5FU was used as median 8th line therapy. All pts had extensive bony disease and 6 had liver involvement. Median PSA at start of treatment was 356 ng/dL. 5 pts progressed within a month of initiation of therapy, and 2 pts did not respond. 8 (61%) patients had treatment for &gt;1 month with 6 having &gt;30% PSA reduction, 4 with a &gt;50% PSA reduction and 2 with a &gt;90% PSA reduction (Table). Median time on therapy was 4.5 months with 2 patients remaining on treatment for &gt;6 months despite multiple pretreatments and far advanced disease. 5FU was well tolerated with improvement in both performance status and symptoms in several patients. Conclusions: Continuous 5FU is active in select highly pretreated pts and targets folate metabolism via inhibition of thymidylate synthase. It is one of the few chemotherapies that can be administered with little myelosuppression, an important issue in far-advanced pts. Prostate specific membrane antigen (PSMA) expression is upregulated in CRPC, especially after abi/enza treatment. PSMA hydrolyzes polyglutamated folates and functions as a folate transporter; its over-expression stimulates both folate transport and prostate cancer cell line growth. We hypothesize that 5FU, functioning as an anti-folate, antagonizes the downstream effects of PSMA over-expression. PSA data for pts on treatment &gt; 1 month. [Table: see text]

Addition of Exogenous γ-Glutamyl Hydrolase Eliminates the Need for Lengthy Incubation of Whole-Blood Lysate for Quantitation of Folate Vitamers by High-Performance Liquid Chromatography–Tandem Mass Spectrometry

BackgroundMeasurement of folate monoglutamates by HPLC–tandem mass spectrometry (HPLC-MS/MS) in whole-blood lysate (WBL) requires lengthy incubation before analysis, risking degradation of labile folate vitamers. ObjectiveWe explored whether the addition of a commercially available recombinant exogenous γ-glutamyl hydrolase (exoGGH) enzyme reduced the required incubation time of WBL for measurement of folate as monoglutamates. MethodsFor conventional deglutamylation of polyglutamates, WBL was incubated for 4 h at 37°C. Alternatively, we added exoGGH to WBL at varying concentrations (1–10 µg/mL) and incubation times (0–90 min). We also investigated modifications to the sample diluent (pH, ascorbic acid compared with sodium ascorbate, and ascorbate concentration). Finally, we tested the effect of the enzyme in different sample types: WBL from frozen whole blood compared with frozen WBL or with frozen washed RBCs. Samples (n ≤ 15/experiment) were analyzed by HPLC-MS/MS for 6 folate monoglutamates and 5-methyltetrahydrofolate diglutamate. ResultsOptimal deconjugation of folate polyglutamates was achieved by using 1% ascorbic acid and 5 µg enzyme/mL WBL, requiring ≤30 min incubation time to achieve complete folate recovery as monoglutamates. This treatment resulted in similar folate concentrations as conventional deglutamylation (4 h at 37°C). The exoGGH enzyme was effective in samples stored frozen as whole blood and as WBL. However, the extended thaw time of whole blood resulted in 5-methyltetrahydrofolate loss and unacceptable changes to the non-methyl folate concentration. Total folate (with exoGGH) measured in washed RBCs was ∼15% lower than RBC folate calculated from WBL concentrations (conventional deglutamylation). ConclusionsThe use of exoGGH minimized incubation time and thus may avoid degradative losses of labile folate forms during sample preparation. The lower folate results in washed RBCs may be due to inadequate packing of RBCs, among other unidentified factors. A larger study is required to confirm the lack of differences in folate concentrations determined with and without the use of exoGGH.

Folate depletion and increased glutamation in juvenile idiopathic arthritis patients treated with methotrexate.

Folates exist as a fluctuating pool of polyglutamated metabolites that may serve as a clinical marker of methotrexate (MTX) activity. This study was undertaken to evaluate circulating folate content and folate polyglutamate distribution in juvenile idiopathic arthritis (JIA) patients and in a cell culture model based on MTX exposure and folate supply. Blood, plasma, and red blood cell (RBC) measurements of MTX and folates were obtained from previously published data sets and an additional analysis of JIA patients receiving MTX (n = 98) and those not receiving MTX (n = 78). Erythroblastoid cells maintained in culture were exposed to MTX and grown under varying levels of folic acid supplementation. Samples were analyzed for cellular folate and MTX content. Circulating folate levels were lower in JIA patients receiving MTX, with reduced levels of blood, plasma, and RBC 5-methyl-tetrahydrofolate (5mTHF) (P < 0.0001). Average polyglutamate chain length (Gluavg ) of RBC 5mTHF was elevated in JIA patients receiving MTX (median ± interquartile range 5.63 ± 0.15 versus 5.54 ± 0.11 in those not receiving MTX; P < 0.001) and correlated with both RBC MTX accumulation (P = 0.02) and reduced plasma 5mTHF levels (P = 0.008). MTX exposure and folate deprivation in erythroblastoid cells resulted in a depletion of bioactive folate species that was associated with a shift to higher Gluavg values for several species, most notably tetrahydrofolate (THF) and 5,10-methylene-tetrahydrofolate (CH2 THF). Increased Gluavg resulted from the depletion of short-chain and the accumulation of long-chain glutamate species. Our findings indicate that folate content and polyglutamate distribution are responsive markers of MTX activity and folate supply in vivo and in vitro, and may provide novel clinical markers of pharmacologic activity of MTX.

Structures of Plasmodium vivax serine hydroxymethyltransferase: implications for ligand-binding specificity and functional control.

Plasmodium parasites, the causative agent of malaria, rely heavily on de novo folate biosynthesis, and the enzymes in this pathway have therefore been explored extensively for antimalarial development. Serine hydroxymethyltransferase (SHMT) from Plasmodium spp., an enzyme involved in folate recycling and dTMP synthesis, has been shown to catalyze the conversion of L- and D-serine to glycine (Gly) in a THF-dependent reaction, the mechanism of which is not yet fully understood. Here, the crystal structures of P. vivax SHMT (PvSHMT) in a binary complex with L-serine and in a ternary complex with D-serine (D-Ser) and (6R)-5-formyltetrahydrofolate (5FTHF) provide clues to the mechanism underlying the control of enzyme activity. 5FTHF in the ternary-complex structure was found in the 6R form, thus differing from the previously reported structures of SHMT-Gly-(6S)-5FTHF from other organisms. This suggested that the presence of D-Ser in the active site can alter the folate-binding specificity. Investigation of binding in the presence of D-Ser and the (6R)- or (6S)-5FTHF enantiomers indicated that both forms of 5FTHF can bind to the enzyme but that only (6S)-5FTHF gives rise to a quinonoid intermediate. Likewise, a large surface area with a highly positively charged electrostatic potential surrounding the PvSHMT folate pocket suggested a preference for a polyglutamated folate substrate similar to the mammalian SHMTs. Furthermore, as in P. falciparum SHMT, a redox switch created from a cysteine pair (Cys125-Cys364) was observed. Overall, these results assert the importance of features such as stereoselectivity and redox status for control of the activity and specificity of PvSHMT.

Mammalian Mitochondrial and Cytosolic Folylpolyglutamate Synthetase Maintain the Subcellular Compartmentalization of Folates

Folylpoly-γ-glutamate synthetase (FPGS) catalyze the addition of multiple glutamates to tetrahydrofolate derivatives. Two mRNAs for the fpgs gene direct isoforms of FPGS to the cytosol and to mitochondria in mouse and human tissues. We sought to clarify the functions of these two compartmentalized isoforms. Stable cell lines were created that express cDNAs for the mitochondrial and cytosolic isoforms of human FPGS under control of a doxycycline-inducible promoter in the AUXB1 cell line. AUXB1 are devoid of endogenous FPGS activity due to a premature translational stop at codon 432 in the fpgs gene. Loss of folates was not measurable from these doxycycline-induced cells or from parental CHO cells over the course of three CHO cell generations. Likewise, there was no detectable transfer of folate polyglutamates either from the cytosol to mitochondria, or from mitochondria to the cytosol. The cell line expressing cytosolic FPGS required exogenous glycine but not thymidine or purine, whereas cells expressing the mitochondrial isoform required exogenous thymidine and purine but not glycine for optimal growth and survival. We concluded that mitochondrial FPGS is required because folate polyglutamates are not substrates for transport across the mitochondrial membrane in either direction and that polyglutamation not only traps folates in the cytosol, but also in the mitochondrial matrix.

Determination of Total Folates in Infant Formula and Adult Nutritionals by Trienzyme Extraction and UPLC-MS/MS Quantitation: First Action 2011.06

The method for "Determination of Total Folates in Infant Formula and Adult Nutritionals by Trienzyme Extraction and UPLC-MS/MS Quantitation" was submitted to the Folate Working Group for consideration for adoption as Official First Action by AOAC INTERNATIONAL. This method uses ultra performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) to determine the total folates in infant formulas and adult nutritionals after trienzyme digestion. Deconjugation of the various folate polyglutamates to folate monoglutamates is achieved by using rat plasma conjugase after the sample digestion with protease and a-amylase during the trienzyme digestion process. This method shows linearity of folate concentrations in the range of 10-19 100 microg/100 g. Extension of the range to cover folate concentrations of 5-2 000 000 microg/100 g can be achieved with appropriate adjustment of the sample weight and SPE cleanup loading volume. The recoveries ranged from 94.10 to 101.34%.

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

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. 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. 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. 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.

Glutamate: a truly functional amino acid

Glutamate is one of the most abundant of the amino acids. In addition to its role in protein structure, it plays critical roles in nutrition, metabolism and signaling. Post-translational carboxylation of glutamyl residues increases their affinity for calcium and plays a major role in hemostasis. Glutamate is of fundamental importance to amino acid metabolism, yet the great bulk of dietary glutamate is catabolyzed within the intestine. It is necessary for the synthesis of key molecules, such as glutathione and the polyglutamated folate cofactors. It plays a major role in signaling. Within the central nervous system, glutamate is the major excitatory neurotransmitter and its product, GABA, the major inhibitory neurotransmitter. Glutamate interaction with specific taste cells in the tongue is a major component of umami taste. The finding of glutamate receptors throughout the gastrointestinal tract has opened up a new vista in glutamate function. Glutamate is truly a functional amino acid.

Red blood cell folate concentrations and polyglutamate distribution in juvenile arthritis

Methotrexate (MTX) has several enzymatic targets in the folate pathway. To better understand the variability in response to MTX, we characterized the interindividual variability of intracellular folate pools in children with juvenile arthritis (JA) and determined clinical and genetic contributors to this variability. This exploratory single-center cross-sectional study evaluated 93 patients with JA not currently receiving MTX. Whole blood, plasma, and erythrocyte folate concentrations were determined after deconjugation and analyzed through reversed-phase separation and stable isotope dilution tandem mass spectrometry. Folate polyglutamates were measured in red blood cell lysates using an ion-pair reversed phase chromatography tandem mass spectrometry method. Intracellular concentrations of 5-methyl-tetrahydrofolate (5-CH3-THF) and 5,10-methenyl-tetrahydrofolate varied approximately 20-fold and 80-fold, respectively. The polyglutamated forms of 5-CH3-THF as a percentage of total 5-CH3-THF (5-CH3-THFGlun) were also measured. Hierarchical clustering of 5-CH3-THFGlun revealed two groups, each with two distinct clusters. There was an inverse relationship between 5-CH3-THFGlun chain length and plasma 5-CH3-THF concentrations. A subgroup of patients with a historical intolerance to MTX had significantly lower cellular folate concentrations (P<0.0001). In univariate analyses, clinical variables including sex, age, and folate supplementation in addition to variations in MTHFR, MTR, and SLC25A32 were associated with differential intracellular folate redox concentrations. Multivariate analysis further supported the association of single nucleotide polymorphisms in SLC25A32, MTHFR, and MTR with variability in intracellular 5-CH3-THF and 5,10-methenyl-tetrahydrofolate concentrations, respectively. Measurement of intracellular folate isoforms may contribute toward a better understanding of individual MTX effects in JA. Clinical variables in addition to genotypic differences beyond MTHFR may additionally explain differential intracellular folate concentrations and variable responses to MTX.

Alcohol-associated folate disturbances result in altered methylation of folate-regulating genes

Folate plays a critical role in maintaining normal metabolic, energy, differentiation and growth status of all mammalian cells. The steady-state accumulation of folate seems to depend on the activity of two enzymes: folylpolyglutamate synthetase (FPGS), which adds glutamate residues, and gamma-glutamyl hydrolase (GGH), which removes them, enabling it to be transported across the biological membranes. Overexpression of GGH and downregulation of FPGS would be expected to decrease intracellular folate in its polyglutamylated form, thereby increasing efflux of folate and its related molecules, which might lead to resistance to drugs or folate deficiency. The study was sought to delineate the activity of GGH and expression FPGS in tissues involved in folate homeostasis during alcoholism and the epigenetic regulation of these enzymes and transporters regulating intracellular folate levels. We determined the activity of GGH and expression of FPGS in tissues after 3 months of ethanol feeding to rats at 1 g/kg body weight/day. The results showed that there was not any significant change in the activity of folate hydrolyzing enzyme GGH in ethanol-fed rats while there was significant down regulation in the expression of FPGS. Ethanol feeding decreased the total as well as polyglutamated folate levels. There was tissue-specific hyper/hypo methylation of folate transporter genes viz. PCFT and RFC by chronic ethanol feeding. Moreover, hypermethylation of FPGS gene was observed in intestine and kidney without any change in methylation levels of GGH in the ethanol-fed rats. In conclusion, the initial deconjugation of polyglutamylated folate by GGH was not impaired in ethanol-fed rats while the conversion of monoglutamylated folate to polyglutamylated form might be impaired. There was tissue-specific altered methylation of folate transporter genes by chronic ethanol feeding.

The Folate Hydrolase 1561C&gt;T Polymorphism Is Associated With Depressive Symptoms in Puerto Rican Adults

To examine the associations between variants of genes involved in the uptake, retention, and metabolism of folate and depressive symptoms and to analyze whether such associations are direct or through mediation by folate or homocysteine. We performed a cross-sectional analysis of data from 976 Puerto Rican adults, aged 45 to 75 years, residing in the greater Boston area, Massachusetts. Twelve single nucleotide polymorphisms (SNPs) in genes involved in folate uptake, retention, and metabolism were investigated. These include FOLH1 (folate hydrolase), FPGS (folate polyglutamate synthase), GGH (γ-glutamyl hydrolase), MTHFR (methylenetetrahydrofolate reductase), MTR (methionine synthase), PCFT (proton-coupled folate transporter), and RFC1 (reduced folate carrier 1). The Center for Epidemiologic Studies Depression Scale (CES-D) was used to measure depressive symptoms. The FOLH1 rs61886492 C>T (or 1561C>T) polymorphism was significantly associated with lower CES-D score (p = .0025) after adjusting for age, sex, population admixture, smoking, and educational attainment. Individuals with the TT and TC genotypes were 49% less likely (odds ratio = 0.51, 95% confidence interval = 0.29-0.89) to report mild depressive symptoms (CES-D score ≥16 and ≤26) and 64% less likely (odds ratio = 0.36, 95% confidence interval = 0.18-0.69) to report moderate to severe depressive symptoms (CES-D score >26), compared with those with the CC genotype. No significant mediation effects by plasma folate or homocysteine on the associations between this single nucleotide polymorphism and CES-D score were observed. The FOLH1 1561C>T polymorphism may be associated with the risk of depressive symptoms.

A central role for gamma-glutamyl hydrolases in plant folate homeostasis

Most cellular folates carry a short poly-γ-glutamate tail, and this tail is believed to affect their efficacy and stability. The tail can be removed by γ-glutamyl hydrolase (GGH; EC 3.4.19.9), a vacuolar enzyme whose role in folate homeostasis remains unclear. In order to probe the function of GGH, we modulated its level of expression and subcellular location in Arabidopsis plants and tomato fruit. Three-fold overexpression of GGH in vacuoles caused extensive deglutamylation of folate polyglutamates and lowered the total folate content by approximately 40% in Arabidopsis and tomato. No such effects were seen when GGH was overexpressed to a similar extent in the cytosol. Ablation of either of the major Arabidopsis GGH genes (AtGGH1 and AtGGH2) alone did not significantly affect folate status. However, a combination of ablation of one gene plus RNA interference (RNAi)-mediated suppression of the other (which lowered total GGH activity by 99%) increased total folate content by 34%. The excess folate accumulated as polyglutamate derivatives in the vacuole. Taken together, these results suggest a model in which: (i) folates continuously enter the vacuole as polyglutamates, accumulate there, are hydrolyzed by GGH, and exit as monoglutamates; and (ii) GGH consequently has an important influence on polyglutamyl tail length and hence on folate stability and cellular folate content.

Severe toxicity of capecitabine following uncomplicated treatment with 5-fluorouracil/leucovorin

Colorectal carcinomas are among the most common tumor types and are generally treated with palliative chemotherapy in case of metastatic disease. Here, we describe the case of a 58 year old woman with metastatic rectal carcinoma who developed severe gastrointestinal toxicity when the thus far well-tolerated intravenous 5-fluorouracil (5-FU)/leucovorin containing chemotherapeutic regimen was replaced by the same chemotherapeutic regimen in combination with the oral 5-FU prodrug capecitabine. This increased toxicity is probably due to the intracellular retention of polyglutamated folates induced by prior leucovorin therapy which, upon subsequent administration of capecitabine, will result in an enhanced and prolonged inhibition of the, for DNA synthesis important, enzyme thymidylate synthase, essentially creating a situation equivalent to overdosing.

Abstract 79: The compartmentalization of folate metabolism depends on two isoforms of folylpoly-γ-glutamate synthetase and the mitochondrial folate transporter

Abstract The reactions of folate metabolism are distributed between the cytosolic and mitochondrial compartments of mammalian cells and both components are essential for mammalian cell survival. The enzyme folylpoly-γ-glutamate synthetase (FPGS) has been shown to be essential for the intracellular retention of folates as polyglutamate derivatives. The fpgs gene produces two transcripts, one for an isoform with a leader peptide targeting it to the mitochondrial matrix, and the other is transcribed without this sequence, so that the encoded isoform remains in the cytosol. Transfer of folates into the mitochondrial matrix is mediated by an inner mitochondrial membrane transport protein, the mitochondrial folate transporter (MFT). Either deletion of the MFT or of mitochondrial FPGS is cytotoxic and renders the cell auxotrophic for glycine; the mechanism of this effect is far from apparent. To better understand this compartmentalization, we generated cell lines that express only the cytosolic or mitochondrial isoform of FPGS under tetracycline-inducible control in FPGS-null AuxB1 cells. Western blot analysis of the FPGS protein in these cell lines indicate that both FPGS isoforms are expressed near endogenous levels in the presence of doxycycline and that each cell line solely expresses the FPGS isoform in the respective compartment. Clonogenic survival of each doxycycline-induced cell line demonstrates a cellular requirement for each FPGS isoform. Additional studies showed that cells complemented with cytosolic FPGS only accumulated and retained folates in the cytosol but not mitochondria, leading us to conclude that folate polyglutamates are not substrates for the MFT. In addition, expression of only the mitochondrial form of FPGS resulted in cells that progressively trapped folates in the mitochondria, leading us to the concept that folate polyglutamates do not escape the mitochondria. However, cytosolic folates were observed in cells expressing only mitochondrial FPGS. We are currently using these cell lines to study the transfer of folates across the mitochondrial membrane and polyglutamation in each compartment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 79.