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PubMed1989

Methenyltetrahydrofolate synthetase prevents the inhibition of phosphoribosyl 5-aminoimidazole 4-carboxamide ribonucleotide formyltransferase by 5-formyltetrahydrofolate polyglutamates.

Methenyltetrahydrofolate synthetase (EC 6.3.3.2) catalyzes the irreversible ATP and Mg2+-dependent transformation of 5-formyltetrahydrofolate (N5-HCO-H4-pteroylglutamic acid (PteGlu] to 5,10-methenyltetrahydrofolate. The physiological function of this reaction remains unknown even though it is potentially involved in the intracellular metabolism of the large doses of N5-HCO-H4-PteGlu (leucovorin) administered to cancer patients. We have tried to elucidate methenyltetrahydrofolate synthetase's physiological role by examining the consequences of its inhibition in MCF-7 human breast cancer cells by the folate analog 5-formyltetrahydrohomofolate (fTHHF), a potent competitive inhibitor with a Ki of 1.4 microM. fTHHF inhibited MCF-7 cell growth with an IC50 of 2.0 microM during 72-h exposures, and this effect was fully reversible by hypoxanthine but not thymidine, indicating specific inhibition of de novo purine synthesis. A correlation was observed between increases in intracellular N5-HCO-H4-PteGlu concentrations following fTHHF and cell growth inhibition. De novo purine synthesis was inhibited at the second folate-dependent enzyme, phosphoribosyl aminoimidazole-carboxamide formyltransferase (AICAR transferase; EC 2.1.2.3), as determined by aminoimidazole carboxamide rescue and azaserine inhibition studies. N5-HCO-H4-PteGlu pentaglutamate was a potent inhibitor of purified MCF-7 cell AICAR transferase with a Ki of 3.0 microM while the monoglutamate was not an inhibitor up to 10 microM and fTHHF was only weakly inhibitory with a Ki of 16 microM. These findings suggest that methenyltetrahydrofolate synthetase activity is needed to prevent de novo purine synthesis inhibition by N5-HCO-H4-PteGlu polyglutamates.

Acyltransferases↗
PubMed1993

The properties and function of gamma-glutamyl hydrolase and poly-gamma-glutamate.

gamma-Glutamyl hydrolase is a ubiquitous enzyme that has the capacity to cleave gamma-glutamyl bonds of cellular folyl- and antifolylpoly-gamma-glutamates. This study has revealed that the enzyme is secreted by primary cultures of rat hepatocytes and by H35 hepatoma cells. It was found that more than 99% of the total enzyme from H35 cells accumulated in the medium after 48 hr incubation with the serum-free medium. The cells were shown to remain intact during the secretion period since lactate dehydrogenase, dihydrofolate reductase and lysosomal hydrolases other than gamma-glutamyl hydrolase were retained within the cell. When PteGlu5 (folylGlu4) is used as a substrate the initial product is PteGlu (folate), and there is no appearance of intermediate chain length pteroyl polyglutamates. Therefore, the secreted and cellular gamma-glutamyl hydrolase from hepatoma cells appears to be an endopeptidase. Polyclonal antibodies to the poly-gamma-glutamate substrates of the enzyme were prepared and characterized. The antibodies recognize the structural differences between alpha- and gamma-glutamyl linkages but appear equally active with PteGlu5 and its analogs such as 4-NH2-10-CH3PteGlu5 and pABAGlu5. The affinity of the antibodies is related to the gamma-glutamyl structure since L-glutamic acid, folate or p-aminobenzoic acid are inactive with the antibodies. Furthermore, poly-gamma-glutamate has lower affinity for the antibodies than the poly-gamma-glutamate derivatives of PteGlu, 4-NH2-10-CH3PteGlu or pABA.

Animals↗
PubMed1985

Enhanced inhibition of thymidylate synthase by methotrexate polyglutamates.

We have studied the effects of methotrexate (MTX-Glu1) and the polyglutamate derivatives of methotrexate (MTXPGs) with 2, 3, 4, and 5 glutamyl residues on the catalytic activity of thymidylate synthase purified from MCF-7 human breast cancer cells and on the kinetics of the ternary complex formation by 5-fluoro-2'-deoxyuridine 5'-monophosphate, folate cofactor, and thymidylate synthase. MTX-Glu1 exhibited uncompetitive inhibition of thymidylate synthase when reaction kinetics were analyzed by either double reciprocal plots or a computerized mathematical model based on nonlinear least-squares curve fitting. The Ki for MTX-Glu1 inhibition was 13 microM and the I50 was 22 microM, irrespective of the degree of polyglutamation of the folate. In contrast, the polyglutamated derivatives of MTX all acted as noncompetitive inhibitors. The MTXPGs had 75-300-fold greater potency than MTX-Glu1 as inhibitors of thymidylate synthase catalytic activity, with Ki values from 0.17 to 0.047 microM for MTX-Glu2 to MTX-Glu5, respectively. Neither MTX-Glu1 nor MTXPGs promoted the formation of a charcoal-stable ternary complex with thymidylate synthase and 5-fluoro-2'-deoxyuridine 5'-monophosphate. CH2-H4PteGlu5 (where PteGlu represents pteroylglutamic acid) was found to be 40-fold more potent than CH2-H4PteGlu1 in participating in the formation of a ternary complex, and 10 microM MTX-Glu5 significantly inhibited the formation of a ternary complex containing this folate as cofactor. The inhibition was determined to be due to a reduction in the kon. The potency of this inhibition was markedly greater in the presence of CH2-H4PteGlu1 as compared to CH2-H4PteGlu5. This finding suggests that the degree of interference with complex formation in intact cells would depend on the state of polyglutamation of available folate cofactor. Ternary complex formation with H2PteGlu5 as the folate cofactor was also investigated, and a 50% reduction in complex formation was found in the presence of a 2 microM concentration of MTX-Glu5. These findings have significant implications regarding the mechanism of action of MTX-Glu1 and contribute to an understanding of the complex interactions of MTX-Glu1 and 5-fluorouracil.

Breast Neoplasms↗
PubMed1993

Specific and sensitive high-performance liquid chromatographic method with fluorescence detection for measurement of lometrexol and its polyglutamates in biologic samples.

A reversed-phase high-performance liquid chromatographic (HPLC) assay is described for the quantitative determination of lometrexol in biological samples; the assay is rapid, simple, specific, and highly sensitive. The method requires the dissociation of lometrexol from folate-binding proteins present in blood and formation of a fluorescent oxidized derivative of the compound. The dissociation of lometrexol from folate-binding proteins was achieved by acidification to pH 3.5 using ammonium formate, followed by serum protein precipitation with perchloric acid. The protein-free lometrexol was subsequently oxidized by MnO2 at 90 degrees C for 10 min. Chromatographic separation of lometrexol without interference was achieved on a C18 reversed-phase column with a convex gradient, using acetonitrile-0.1% ammonium formate, pH 7.0, as the mobile phase. In human serum and urine the calibration curve was linear between 5 and 300 nM. The lower limit of quantification was 5 nM. The method has been applied successfully to measure serum and urinary levels of lometrexol in patients.

Chromatography, High Pressure Liquid↗
PubMed2000

Electrostatic core shielding in dendritic polyglutamic porphyrins.

Polyglutamic dendritic porphyrins of the general formula H2PophGlu(N)OR (H2Porph = free-base meso-tetra-4-carboxyphenylporphyrin (H2TCPP), Glu=dendrimer layer composed of L-glutamates, N= 1-3: dendrimer generation number, R = terminal group (All, H)) were synthesized and characterized with NMR and MALDI-TOF mass spectroscopy. The free-acid terminated compounds were found to be highly soluble in water, with both their absorption and fluorescence spectra dependent on pH. The value of the porphyrin mono-protonation constant, measured by fluorescence rationing, increased monotonously in the studied series of dendrimers (pK3=6.31. 6.70, and 6.98, for N=1, 2, 3, respectively). For the largest dendrimer, H2PorphGlu(3)OH, pK3 was found shifted by almost two pH units relative to the non-modified H2Porph. The second protonation constant (K4) was much less affected by the dendritic substituents. At pH values less than 3.5 there were noticeable changes in fluorescence intensity and quantum yield even for the highly soluble H2PorphGlu(3)OH. This suggests that interactions between individual dendritic molecules in solution are favored by full protonation of the peripheral glutamic carboxyls. The "dendrimer-protected" porphyrins are convenient fluorescent pH sensors in the biological pH range.

Absorption↗
PubMed1991

The biochemical pharmacology of the thymidylate synthase inhibitor, 2-desamino-2-methyl-N10-propargyl-5,8-dideazafolic acid (ICI 198583).

2-Desamino-2-methyl-N10-propargyl-5,8-dideazafolic acid (ICI 198583) is a more water-soluble analogue of the quinazoline-based thymidylate synthase (TS) inhibitor, N10-propargyl-5,8-dideazafolic acid (CB3717). A 3-fold loss in TS inhibitory activity (murine and human TS, Ki = 10 nM) was accompanied by a 40-fold increase in growth inhibitory potency against L1210 and W1L2 cells in vitro (IC50 = 0.085 and 0.05 microM, respectively) when compared with CB3717. In L1210 cells a concentrative uptake mechanism was demonstrated for [3H]ICI 198583 (Kt = 2.9 microM). The L1210:1565 cell line, with an impaired ability to transport reduced folates or methotrexate (MTX), was resistant (100-fold relative to the wild-type L1210 line) to ICI 198583 (but not CB3717) and did not take up [3H]ICI 198583 significantly. The measurement of folylpolyglutamate synthetase (FPGS) substrate activity demonstrated a Km of 40 microM for ICI 198583 and a Vmax/Km (relative to folic acid) of 3.5. The formation of intracellular polyglutamate derivatives was demonstrated in both L1210 (mouse) and WIL2 (human) cells grown in vitro after exposure to 1 microM [3H]ICI 198583. In L1210 cells, by 4 hr, approximately 50% of the intracellular 3H(approximately 1 microM) was found as polyglutamate forms of ICI 198583, principally as tri- and tetraglutamates. After 24 hr the ICI 198583 polyglutamate pool had expanded, the tetraglutamate metabolite predominated and there was significant formation of the pentaglutamate. Upon resuspension of L1210 cells in drug free medium, ICI 198583 was largely lost from the cells but the polyglutamates were preferentially retained, after 24 hr approximately 70% remained. Synthetic ICI 198583 polyglutamates were shown to be up to 100-fold more potent as inhibitors of isolated TS than the parent compound. Following in vivo administration (500 mg/kg i.v.) ICI 198583 was cleared rapidly from the plasma of mice (T1/2 beta = 16 min, clearance = 42 mL/min/kg). Despite this clearance there was prolonged, dose-dependent inhibition of TS in L1210:NCI cells in vivo. Thus, following 500 mg/kg i.v. the flux through TS was inhibited by greater than 80% for at least 24 hr. Administration of five doses at 5 mg/kg daily of ICI 198583 to L1210:ICR tumour-bearing mice resulted in greater than 60% of the mice being cured, a 10-fold improvement in potency over CB3717. The maximum tolerated dose (MTD) for ICI 198583 using this schedule was greater than 500 mg/kg/day compared with 200 mg/kg/day of CB3717.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1977

Biosynthesis of folate polyglutamate in the rat with different tracers.

1. The recent suggestions that folate polyglutamate biosynthesis demonstrated in vivo with [3'5'9(n)-3H]folic acid is due to exchange and cannot be achieved with [2(-14)C]folic acid has been demonstrated to be untrue. 2. The compounds formed with both types of radioactive tracer have been shown to be folate polyglutamates from their elution position from precalibrated ion-exchange columns and their susceptibility to hydrolysis by a gamma-carboxypeptidase (conjugase) from human sera. 3. By the use of the methods involving [2(-14)C]folic acid, reported by others to give only folate monoglutamates, we have been able to demonstrate clearly the presence of folate polyglutamates.

Animals↗
PubMed1999

[Methotrexate in therapy of gastrointestinal diseases].

This review is not only a comprehensive synopsis of the published data on methotrexate treatment for inflammatory bowel disease and primary biliary cirrhosis but gives also an overview about acting mechanisms, pharmacologic properties and limiting toxicities of low-dose methotrexate. Methotrexate and its polyglutamate analogues are structurally related to folic acid and inhibit many enzymes in the metabolic pathway of folic acid. Long-term low-dose methotrexate treatment (7.5-25 mg) inhibits production of thymidylate, purines, and methionine and leads to intracellulary accumulation of adenosin. These actions lead to inhibition of cellular proliferation, decreased formation of antibodies and reduced productions of inflammatory medicators. Several trials have demonstrated the efficacy of low-dose methotrexate therapy for certain indications as a promising new agent in the treatment of inflammatory bowel disease and primary biliary cirrhosis. However, several serious toxicities potentially limit its use.

Antimetabolites, Antineoplastic↗
PubMed2003

Transport of methotrexate, methotrexate polyglutamates, and 17beta-estradiol 17-(beta-D-glucuronide) by ABCG2: effects of acquired mutations at R482 on methotrexate transport.

ABCG2 is a plasma membrane efflux pump that is able to confer resistance to several anticancer agents, including mitoxantrone, camptothecins, anthracyclines, and flavopiridol. The antimetabolite methotrexate (MTX) was inferred recently to be an additional substrate of the pump based on the analysis of ABCG2-overexpressing cell lines. However, the transport characteristics of the pump with regard to this agent have not been determined. In addition, physiological substrates of ABCG2 have not been identified. Here we examine the in vitro transport properties of the pump using membrane vesicles prepared from HEK293 cells transfected with ABCG2 expression vector. In so doing it is shown that MTX is a high capacity low affinity substrate of the pump, with K(m) and V(max) values of 1.34 +/- 0.18 mM and 687 +/- 87 pmol/mg/min, respectively. Unlike previously characterized multidrug resistance protein family members, ABCG2 is also able to transport MTX diglutamate and MTX triglutamate. However, addition of even one more glutamyl residue is sufficient to completely abrogate ABCG2-mediated transport. By contrast with the wild-type protein (ABCG2-R482), two ABCG2 variants that have been identified in drug selected cell lines, R482T and R482G, were unable to transport MTX to any extent. Similarly, folic acid was subject to efflux by the wild-type protein but not by the two mutants. However, transport of the reduced folate leucovorin was not detected for either the wild-type or the mutant proteins. Finally, it is shown that ABCG2 is capable of transporting E(2)17betaG with K(m) and V(max) values of 44.2 +/- 4.3 micro M and 103 +/- 17 pmol/mg/min, respectively. These results indicate that ABCG2 is a component of the energy-dependent efflux system for certain folates and antifolates, but that its transport characteristics with respect to polyglutamates and reduced folates are not identical to those of multidrug resistance protein family members. In addition, it is demonstrated that R482 mutations observed in drug-resistant cell lines have profound effects on the in vitro transport properties of the pump.

ATP Binding Cassette Transporter, Subfamily G, Mem↗
PubMed2003

HPLC determination of erythrocyte methotrexate polyglutamates after low-dose methotrexate therapy in patients with rheumatoid arthritis.

BACKGROUND: Methotrexate (MTX) may produce antiarthritic effects through polyglutamation to methotrexate polyglutamates (MTXPGs), a process that covalently attaches sequential gamma-linked glutamic residues to MTX. We sought to develop an innovative HPLC method for the quantification of these metabolites in erythrocytes. METHODS: Two alternative approaches were developed. In the first approach, MTXPGs from 50 micro L of packed erythrocytes were converted to MTX in the presence of plasma gamma-glutamyl hydrolase and mercaptoethanol at 37 degrees C. In the second approach, MTXPG species (up to the hepta order of glutamation) from 100 micro L packed erythrocytes were directly quantified in a single run. In both methods, the MTXPGs were extracted from the biological matrix by a simple perchloric acid deproteinization step with direct injection of the extract into the HPLC. The chromatography used a C(18) reversed-phase column, an ammonium acetate/acetonitrile buffer, and postcolumn photo-oxidation of MTXPGs to fluorescent analytes. RESULTS: Intra- and interday imprecision (CVs) were <10% at low and high concentrations of analytes for both methods. The limit of quantification was 5 nmol/L. In 70 patients with rheumatoid arthritis receiving weekly low-dose MTX, the mean (SD) total MTXPG concentration measured after conversion of MTXPGs to MTX was similar to the total MTXPG concentration calculated from the sum of individual MTXPG species [117 (56) vs 120 (59) nmol/L; r = 0.97; slope = 1.0]. The triglutamate predominated over all other MTXPG species (36% of total), the pentaglutamate was the highest order of glutamation detected, and a stability study revealed no change in the polyglutamation pattern in erythrocytes 48 h after phlebotomy when the specimen was stored at 2-8 degrees C. CONCLUSION: The proposed method for quantification of erythrocyte MTXPGs is rapid, sensitive, and accurate and can be applied to the routine monitoring of MTX therapy.

Antirheumatic Agents↗
PubMed2003

Glutathione synthetase homologs encode alpha-L-glutamate ligases for methanogenic coenzyme F420 and tetrahydrosarcinapterin biosyntheses.

Proteins in the ATP-grasp superfamily of amide bond-forming ligases have evolved to function in a number of unrelated biosynthetic pathways. Previously identified homologs encoding glutathione synthetase, d-alanine:d-alanine ligase and the bacterial ribosomal protein S6:glutamate ligase have been vertically inherited within certain organismal lineages. Although members of this specificity-diverse superfamily share a common reaction mechanism, the nonoverlapping set of amino acid and peptide substrates recognized by each family provided few clues as to their evolutionary history. Two members of this family have been identified in the hyperthermophilic marine archaeon Methanococcus jannaschii and shown to catalyze the final reactions in two coenzyme biosynthetic pathways. The MJ0620 (mptN) locus encodes a tetrahydromethanopterin:alpha-l-glutamate ligase that forms tetrahydrosarcinapterin, a single carbon-carrying coenzyme. The MJ1001 (cofF) locus encodes a gamma-F420-2:alpha-l-glutamate ligase, which caps the gamma-glutamyl tail of the hydride carrier coenzyme F420. These two genes share a common ancestor with the ribosomal protein S6:glutamate ligase and a putative alpha-aminoadipate ligase, defining the first group of ATP-grasp enzymes with a shared amino acid substrate specificity. As in glutathione biosynthesis, two unrelated amino acid ligases catalyze sequential reactions in coenzyme F420 polyglutamate formation: a gamma-glutamyl ligase adds 1-3 l-glutamate residues and the ATP-grasp-type ligase described here caps the chain with a single alpha-linked l-glutamate residue. The analogous pathways for glutathione, F420, folate, and murein peptide biosyntheses illustrate convergent evolution of nonribosomal peptide biosynthesis through the recruitment of single-step amino acid ligases.

Adenosine Triphosphate↗
PubMed2006

Method for the determination of blood methotrexate by high performance liquid chromatography with online post-column electrochemical oxidation and fluorescence detection.

Methotrexate (MTX) has been widely used at low dose for the treatment of different diseases including rheumatoid arthritis. MTX might be present in plasma in free form, and in blood cells in methotrexate polyglutamate (MTXPG). A rapid and sensitive HPLC method was developed for the determination of plasma MTX level, whole-blood MTX level, and whole-blood total MTX (MTX+MTXPG) level. To determine plasma MTX level or whole-blood MTX level, a 0.2-ml aliquot of plasma or whole blood (after a freeze-thaw cycle to break blood cells) was well mixed with 0.8 ml methanol and centrifuged. To determine whole-blood total MTX level, a 0.1-ml aliquot of whole blood (after a freeze-thaw cycle) was mixed with 80 microl ascorbic acid (114 mM) and incubated at 37 degrees C for 2h to enzymatically convert the MTXPG to MTX. Then 20 microl NaOH solution (0.5M) and 0.8 ml methanol were added and mixed well. After centrifugation, a 0.5-ml aliquot of the supernatant was evaporated to dryness and re-dissolved in 0.2 ml hydrochloric acid (10mM). Methylene chloride (0.2 ml) was added and mixed well. After centrifugation, the top aqueous layer was injected to HPLC for analysis. After the MTX was eluted from the HPLC column, it was electrochemically oxidized and detected by a fluorescence detector. Recoveries of spiked MTX at ppb (ng/ml) level were between 87.9 and 118% with within-day relative standard deviation less than 5.2% and day-to-day relative standard deviation less than 9.8%. The limit of detection (LOD) and limit of quantitation (LOQ) of the described method were 1.2 and 2.6 ng/ml, respectively.

Arthritis, Rheumatoid↗
PubMed2003

Activation mechanism of the nuclear chaperone nucleoplasmin: role of the core domain.

Nucleoplasmin (NP) mediates nucleosome assembly by removing basic proteins from sperm chromatin and exchanging them with histones. This function is modulated by phosphorylation of NP at multiple sites. NP is pentameric, each monomer consisting of two domains: a core, which forms a stable ring-like pentamer, and a tail, that holds a polyglutamic tract and the nuclear localization signal. In the present study, we have explored the role of the core domain in the functionality of NP. Despite lacking the poly-Glu region, a putative binding site for basic proteins, the isolated core domain of the hyperphosphorylated protein isolated from eggs of Xenopus laevis is able to bind sperm basic proteins and decondense chromatin, in contrast to the inactive, non-phosphorylated recombinant core. This activity can be reproduced artificially in the recombinant core domain through mutation of putative phosphorylation sites to aspartate, thus mimicking the charge effect of phosphorylation. The mutated residues locate in flexible or loop regions exposed on the "distal face" of the core pentamer, where a short acidic region is also found, indicating that phosphorylation might activate the core domain of NP by generating a strong localized negative potential. Our results show that the phosphorylated core domain of NP is active in chromatin decondensation, thus it could contribute together with the poly-Glu containing tail in displaying a binding surface for sperm basic proteins on the NP pentamer.

Animals↗
PubMed2000

Combined structural and biochemical analysis of the H-T complex in the glycine decarboxylase cycle: evidence for a destabilization mechanism of the H-protein.

The lipoate containing H-protein plays a pivotal role in the catalytic cycle of the glycine decarboxylase complex (GDC), undergoing reducing methylamination, methylene transfer, and oxidation. The transfer of the CH(2) group is catalyzed by the T-protein, which forms a 1:1 complex with the methylamine-loaded H-protein (Hmet). The methylamine group is then deaminated and transferred to the tetrahydrofolate-polyglutamate (H(4)FGlu(n)) cofactor of T-protein, forming methylenetetrahydrofolate-polyglutamate. The methylamine group is buried inside the protein structure and highly stable. Experimental data show that the H(4)FGlu(n) alone does not induce transfer of the methylene group, and molecular modeling also indicates that the reaction cannot take place without significant structural perturbations of the H-protein. We have, therefore, investigated the effect of the presence of the T-protein on the stability of Hmet. Addition of T-protein without H(4)FGlu(n) greatly increases the rate of the unloading reaction of Hmet, reducing the activation energy by about 20 kcal mol(-1). Differences of the (1)H and (15)N chemical shifts of the H-protein in its isolated form and in the complex with the T-protein show that the interaction surface for the H-protein is localized on one side of the cleft where the lipoate arm is positioned. This suggests that the role of the T-protein is not only to locate the tetrahydrofolate cofactor in a position favorable for a nucleophilic attack on the methylene carbon but also to destabilize the H-protein in order to facilitate the unlocking of the arm and initiate the reaction.

Amino Acid Oxidoreductases↗
PubMed2000

Neural tube defects among Mexican Americans living on the US-Mexico border: effects of folic acid and dietary folate.

Populations of Mexican descent have high occurrences of neural tube defects (NTDs). A recent study suggested that folic acid supplements may not protect these populations from NTDs. In a case-control study, the authors investigated the role of folic acid and dietary folate intake in NTD risk among Mexican Americans living along the Texas-Mexico border. From January 1995 to February 1999, 148 Mexican-American women with NTD-affected pregnancies and 158 women with normal live births were interviewed in person about use of vitamin supplements and dietary intakes during a 6-month periconceptional period (from 3 months before conception to 3 months after conception). Daily preconceptional consumption of vitamin supplements containing folic acid was 2.5% in control women and 2.0% in case women (odds ratio = 0.77; 95% confidence interval (CI): 0.19, 3.22). With adjustment for maternal age, education, obesity, and previous stillbirth or miscarriage, the risk estimate was essentially null (odds ratio = 1.12; 95% CI: 0.22, 5.78). Combined folic acid intake from diet and supplements showed only a modest risk reduction for intakes of > or = 1.0 mg per day (adjusted odds ratio = 0.73; 95% CI: 0.31, 1.72). The fact that the primary folic acid exposure was in the form of dietary polyglutamates rather than the more easily absorbed supplemental monoglutamates may explain an apparent decreased effect in this population.

Adult↗
PubMed1971

Folates in plasma and bile of man after feeding folic acid--3H and 5-formyltetrahydrofolate (folinic acid).

During the 1st hr after feeding folic acid-(3)H ((3)H-PteGlu) to fasting human volunteers, plasma S. faecalis and (3)H activity were elevated to an equivalent degree, whereas after this, the (3)H activity exceeded S. faecalis activity, which suggests gradual conversion of folic acid-(3)H to methyltetrahydrofolate-(3)H (5-CH(3)H(4) PteGlu). The increase of L. casei activity exceeded the increase of S. faecalis and (3)H activity, which is consistent with flushing of endogenous methyltetrahydrofolate from the tissues by the administered folic acid-(3)H. Feeding of 5-formyltetrahydrofolate (+/-5CHOH(4)PteGlu) produced a large increase of plasma L. casei activity and only a slight increase of S. faecalis and P. cerevisiae activity, which is consistent with very rapid conversion of folinic acid to methyltetrahydrofolate. Bile folate concentration determined microbiologically was 2.3-9.8 times plasma folate. 40-80% of the bile folate was S. faecalis-active and 20-35% P. cerevisiae-active. Chromatography of bile folates on TEAE-cellulose showed several folates including four tentatively identified as 10-formyltetrahydrofolate (10-CHO-H(4)PteGlu), 10-formylfolate (10-CHO-PteGlu), and/or 10-formyldihydrofolate (10-CHOH(2)PteGlu), methyltetrahydrofolate, and possibly a triglutamate folate. After folate ingestion bile folate concentration increased rapidly. The distribution of bile folates measured by microbiological assay was similar after either folic or folinic acid feeding. Most of the (3)H label of folic acid-(3)H appeared in the biological folates of bile rather than in the folic acid fraction, which shows that the administered folic acid was rapidly transformed to other folates. Folate polyglutamate deconjugating enzyme activity was found to be much less than in serum. Polyglutamates of the type found in yeast were not found in bile. It is suggested that biliary folate may reflect the hepatic intracellular oligoglutamate folate pool rather than the folate as it appears in the hepatic portal blood.

Bile↗
PubMed1977

Identification of folate binding proteins in rat liver.

Intraperitoneal injection of tritiated folic acid (PteGlu) into rats has revealed the presence of three separate protein fractions in the cytosol fraction of the liver and one in the mitochondria which bind folate derivatives. The proteins in the cytosol (cytosol I, II and III) have approximate molecular weights of 350,000, 150,000, and 25,000 and the protein in the mitochondria has an approximate molecular weight of 90,000 as estimated by gel filtration. The bound folate derivatives are primarily polyglutamate forms while cytosol II contains primarily bound 5-methyltetrahydrofolate polyglutamate derivatives. Little binding of radioactively labeled folic acid or 5-methyltetrahydrofolate to these fractions was observed when binding was carried out in vitro. Significant binding in vitro was observed, however, when a mixture of biosynthetically labeled natural folate derivatives was used. These proteins have not been purified, but cytosol III partially consists of the enzyme, tetrahydrofolate dehydrogenase (EC 1.5.1.3). Studies on the time course of folic acid incorporation into the liver showed that soon after injection nonmetabolized folic acid was bound to the plasma membrane fraction of the liver cell. It is suggested that at least one of the binding proteins in the cytosol may be involved in storage of the vitamin while the binding of nonmetabolized folic acid to the plasma membrane may reflect the existence of a carrier for folic acid transport into the cell.

Animals↗