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PubMed1979

Effect of anticonvulsant drugs on the rate of folate catabolism in mice.

An increase in folate catabolism has been suggested as the cause of the folate deficiency observed in many clinical conditions, including chronic anticonvulsant therapy. Previous studies have shown that the radioactive catabolites, excreted after an equilibration period of 3 d, consisted exclusively of folates that had been cleaved to produce pteridines and p-aminobenzoylglutamate, most of which was excreted as acetamidobenzoylglutamate. We have developed an experimental animal model using mice to determine the rate of catabolism of [3H]pteroylglutamate (folic acid) by the quantitative estimation of [3H]p-aminobenzoylglutamate and [3H]acetamidobenzoylglutamate in urine. Administration of diphenylhydantoin at three different doses (0.5, 20, and 50 mg/kg) significantly increased the rate of catabolism as measured by an increase in both the mean daily excretion and the cumulative excretion of these catabolites. Administration of intramuscular phenobarbitone on the other hand, did not affect the rate of catabolism, when compared with controls.

Animals↗
PubMed1999

Thymidylate synthase level as the main predictive parameter for sensitivity to 5-fluorouracil, but not for folate-based thymidylate synthase inhibitors, in 13 nonselected colon cancer cell lines.

Thymidylate synthase (TS), a critical enzyme in the de novo synthesis of thymidylate, is an important target for fluoropyrimidines and folate-based TS inhibitors. In a panel of 13 nonselected human colon cancer cell lines, we evaluated the role of TS levels in sensitivity to 5-fluorouracil (5FU) and four folate-based TS inhibitors that have been introduced recently into the clinic: ZD1694 (Tomudex, Raltitrexed, TDX), GW1843U89 (GW), LY231514 (LY), and AG337 (Thymitaq, AG). Because the latter compounds have different transport and polyglutamylation characteristics, we also related these parameters with drug sensitivity, measured by the sulforhodamine B assay after 72 h of drug exposure. For 5FU, the IC50s varied from 0.8 to 43.0 microM. Leucovorin (LV) potentiated the activity of 5FU in only 4 of 13 cell lines. Sensitivity to folate-based TS inhibitors was variable; IC50s were in the range of: 5.3-59.0 nM TDX; 11.0-1570 nM LY; and 0.5-8.9 nM GW. Eleven of 13 cell lines had an IC50 for AG between 1.3 and 5.3 microM. Two cell lines were resistant to AG, Colo201 and SW1116, with IC50s of 27 and 29 microM, respectively. TS catalytic activity (conversion of dUMP to dTMP) varied from 62 to 777 pmol/h/10(6) cells. The number of FdUMP binding sites varied from 32 to 231 fmol/10(6) cells. Regression analysis showed a significant relation between TS catalytic activity and IC50s for 5FU and 5FU/LV. Kis for FdUMP showed a significant Spearman rank correlation with the IC50s of AG and GW. The role of antifolate transport, accumulation, and polyglutamylation was determined with [3H]methotrexate (MTX) as a reference compound. [3H]MTX influx via the reduced folate carrier varied from 18.6 to 150 fmol/10(6) cells/min. Folylpolyglutamate synthetase (FPGS) activity showed a range from 47 to 429 pmol/10(6) cells/h. A total of 24 h of [3H]MTX accumulation showed a 20-fold variation, from 1.2 to 21.8 pmol/10(6) cells. FPGS levels showed a Spearman rank positive correlation with cytotoxicity to TDX. In conclusion, in a heterogeneous nonselected human colon cancer cell line panel, the best predictor for sensitivity to 5FU and 5FU/LV was TS activity. Multiple sensitivity determinants were of importance for antifolate TS inhibitors, including FPGS activity and TS enzyme kinetics.

Antimetabolites, Antineoplastic↗
PubMed1990

The folate cofactor of Escherichia coli DNA photolyase acts catalytically.

Escherichia coli DNA photolyase catalyzes the light-driven (300-500 nm) repair of pyrimidine dimers formed between adjacent pyrimidine bases in DNA exposed to UV light (200-300 nm). The light-driven repair process is facilitated by two enzyme-bound cofactors, FADH2 and 5,10-methenyltetrahydrofolate. The function of the folate has been characterized in greater detail in this series of experiments. Investigations of the relative binding affinities of photolyase for the monoglutamate and polyglutamate forms of 5,10-methenyltetrahydrofolate show that the enzyme has a greater affinity for the naturally occurring polyglutamate forms of the folate and that the exogenously added monoglutamate derivative is less tightly associated with the protein. Multiple turnover experiments reveal that the folate remains bound to photolyase even after 10 turnovers of the enzyme. Examination of the rates of repair by photolyase containing stoichiometric folate in the presence or absence of free folate under multiple turnover conditions and at micromolar concentrations of enzyme also demonstrates that the folate acts catalytically. The stimulation of turnover by exogenous folate seen at low concentrations of photolyase is shown to be due to the lower affinity of photolyase for the monoglutamate derivative used in reconstitution procedures. These results demonstrate that the folate of E. coli DNA photolyase is a bona fide cofactor and does not decompose or dissociate during multiple turnovers of the enzyme.

Catalysis↗
PubMed1979

Differential inhibition of host and viral thymidylate synthetases by folylpolyglutamates.

The ability of folate analogues to inhibit host and viral thymidylate synthetases was measured using the corresponding Escherichia coli and T2-phage-induced enzymes. In the absence of Mg2+, 6 x 10(-7) M pteroylhexaglutamate inhibited the T2-phage-induced synthetase by 50%, but at least 100-fold greater levels of this compound were necessary to inhibit the E. coli synthetase by this amount. At 2.5 x 10(-6) M pteroylhexaglutamate, at least 80% inhibition of the T2-phage synthetase could be obtained with little or no inhibition of the E. coli enzyme. The pteroylmonoglutamate was about 2 orders of magnitude less inhibitory towards the T2-phage enzyme than the pteroyltri- to -heptaglutamates. However, upon addition of Mg2+ to the assay mixture, the inhibition produced by pteroylhexaglutamate was essentially reversed, with the E. coli synthetase now increasingly inhibited by this compound and the T2-synthetase only minimally impaired. Methotrexate and N10-formyl-2-amino-4-hydroxyquinazoline, although inhibitory to both enzymes in the presence or absence of Mg2+, did not show this differential selectivity. These results suggest that certain folate analogues may be useful in distinguishing between a host and an infecting organism's thymidylate synthetase and could thus provide an additional means of screening for potential chemotherapeutic agents.

Coliphages↗
PubMed1984

Synthesis and properties of 7-hydroxymethotrexate polyglutamyl derivatives in Ehrlich ascites tumor cells in vitro.

The synthesis of poly-gamma-glutamyl derivatives of 7-hydroxymethotrexate (7-OH-4-NH2-10-CH3-pteroyl-glutamic acid (PteGlu1] was evaluated by direct hydroxylation of the tetraglutamyl derivative of methotrexate (4-NH2-10-CH3-PteGlu4) by a cell-free preparation of rabbit liver aldehyde oxidase and by polyglutamylation of 7-OH-methotrexate in Ehrlich ascites tumor cells in vitro. The polyglutamyl derivatives of 7-OH-methotrexate rapidly accumulate in cells to the 7-OH-4-NH2-10-CH3-PteGlu4. While 7-OH-methotrexate monoglutamate does not bind to dihydrofolate reductase, 7-OH-4-NH2-10-CH3-PteGlu4 does bind to the enzyme as established by gel filtration analysis of cell extracts and by use of purified dihydrofolate reductase from Ehrlich cells. Within cells, the rate of formation of 7-OH-methotrexate polyglutamyl derivatives exceeds that for methotrexate by a factor of 2.7 at comparable free monoglutamyl substrate levels, suggesting that 7-OH-methotrexate may be a better substrate than methotrexate for the folylpolyglutamate synthetase. 7-OH-methotrexate slows the rate of methotrexate polyglutamylation in cells, a consequence of the inhibition of methotrexate transport with reduced methotrexate substrate available for polyglutamylation. When 7-OH-methotrexate polyglutamyl derivatives were accumulated inside the cells following which extracellular 7-OH-methotrexate was removed, the monoglutamate, and to a lesser extent the diglutamate, exited the cells whereas the majority of the longer polyglutamyl derivatives were retained and continued to be metabolized to higher forms. These studies suggest that 7-OH-methotrexate and its polyglutamyl derivatives may play a role in modulating methotrexate action, either by their own inhibitory effects on folate-dependent enzymes or by their effects on methotrexate transport and metabolism within cells.

Aldehyde Oxidase↗
PubMed1982

The metabolism of [3',5',9(n)-3H] pteroylglutamic acid by chicken liver in vivo. Effects of folate deficiency and vitamin B12 supplementation.

1. Control, folate-deficient and vitamin B12-supplemented chickens were given [3', 5', 9 (n)-3H] pteroylglutamic acid by intraperitoneal injection. After various time intervals (1.5 h-8d) the incorporation of radioactivity into hepatic folate derivatives was measured. 2. The results confirm that avian species have a faster assimilation of folate than most mammalian species. 3. Folate deficiency increased the incorporation and retention of [3H] pteroylglutamic acid. The degree of folate conjugation was unaffected by folate deficiency but the formyl to methyl folate ratio was decreased. 4. Vitamin B12 supplementation had no effect on the uptake or conjugation of [3H] pteroylglutamic acid, but increased the formyl to methyl folate ratio.

Animals↗
PubMed1997

The effects of combined antifolates on inhibition of growth of murine leukemia cells cultured in vitro.

The synergistic effect of trimetrexate (TMTX) and sulphonamide derivatives of quinazoline on the cultured 5178Y murine leukemia cells was examined. On exposure to the slightly inhibitory concentrations of TMTX (0.1 nM) in combination with 2-desamino-2-methyl-10-propargyl-5,8-dideaza-pteroyl-sulphoglyc ine (DMPDDSF) (0.02 microM) a synergistic inhibitory effect of the antifolates on cell growth was observed. These two drugs in the same combination caused also synergistic inhibition of de novo synthesis of thymidylate in intact cells as measured by tritium release from [5-(3)H]deoxyuridylate. This was accompanied by a marked reduction in intracellular concentration of 5,10-methylenetetrahydro-pteroyl-polyglutamate (5,10CH2H4PteGlu(n)) (0.2 microM) and dihydropteroyl-polyglutamate (0.12 microM). In these conditions de novo biosynthesis of purine was decreased by 50%. These observations show that growth inhibition by combined antifolates is mediated by intracellular depletion of the substrate of thymidylate synthase -- 5,10CH2H4PteGlu(n). The results obtained strongly suggest that under certain conditions inhibition of thymidylate synthesis by DMPDDSF is intensified by prior application of TMTX -- an inhibitor of dihydrofolate reductase.

Animals↗
PubMed1983

Folate utilization in Friend erythroleukemia cells.

In order to study the generation, factors controlling endogenous folate pools, and their functional importance, Friend erythroleukemia cells were grown in media containing 100; 1,000; and 10,000 ng/ml of tritiated pteroylglutamic acid (3H)PteGlu1 and then studied in unlabeled media with varying amounts of PteGlu1. The intracellular folate pool was directly proportional to the PteGlu1 in which the cells were incubated. At equilibrium, greater than 95% of the labeled intracellular folate pool chromatographed as polyglutamyl folate, regardless of the exogenous folate concentration. The functional importance of the intracellular folate pool was studied by varying the endogenous pool and the exogenous (media) supply. The ability of the cells to replicate in the absence of exogenous folate was directly proportional to the intracellular polyglutamyl folate pool. The maximal rate of replication, however, required exogenous PteGlu1 in addition. The cell doubling time was the most important determinant of intracellular folate turnover; changes in the intracellular pool size and the extracellular folate concentration had no effect on the turnover time. In a rapidly proliferating tissue, the onset of functional folate deficiency will be determined by dilution of intracellular polyglutamates among progeny until a critical level is reached.

Animals↗
PubMed1983

New methods for the study of folate coenzymes: endogenous polyglutamate patterns of subcellular hepatocyte fractions and of regenerating rat liver.

A procedure has been developed that permits the quantitation of three different pools of one-carbon-substituted folates with the simultaneous determination of their corresponding poly-gamma-glutamyl chain lengths. Pool 1 is made up of N5,N10-methylene-tetrahydrofolates and unsubstituted tetrahydro and dihydrofolates. Pool 2 is made up solely of N5-methyl-tetrahydrofolates and pool 3 includes the folates with one-carbon-substituents at the formyl oxidation level: N5,N10-methenyl-tetrahydrofolates, N10- and N5-formyl-tetrahydrofolates, and N5-formimino-tetrahydrofolates. Conditions have been found that permit the selective cleavage of the C9-N10 bond of the folates of pools 1, 1 + 2, and 1 + 2 + 3. The cleaved folates are quantitated as the Bratton-Marshall azo-dyes of the p-amino-benzoyl-poly-gamma-glutamates (AzoGlun) after converting the uncleaved pools to Bratton-Marshall negative products. Determination of the poly-gamma-glutamyl chain length of the cleaved pools is carried out by high pressure liquid chromatography of the AzoGlun comparing the elution position of each peak with that of authentic synthetic markers. Quantitation of the individual peaks is obtained by integration and by reference to an internal synthetic standard (usually AzoGlu3). To increase the sensitivity of the procedure and make it applicable to plasma or needle biopsy specimens we have developed a synthesis for [3H]-N'-naphthylethylenediamine. This compound is the one coupled to the diazotized p-aminobenzoyl-poly-gamma-glutamates to form the AzoGlun in the Bratton-Marshall test. The new methods do not require labeling the animal's folates by the prior injection of radioactive folic acid thus permitting the study of endogenous folate patterns. Applied to rat liver these methods have shown that pool 3 accounts for 44.3 +/- 7.7%, pool 2 for 36.7 +/- 4.7%, and pool 1 for 18.5 +/- 5.8% of the total folates. The pentaglutamates made up about 52% and the hexaglutamates about 42% of the folates. Hepta and tetraglutamates account for approximately 2.8 and 3.5% respectively. The cytosolic folates are predominantly hexaglutamates whereas pentaglutamates predominate in the heavy mitochondrial fraction. Profound changes in the relative proportions of the polyglutamates were observed 24 h after partial hepatectomy. There are marked increases in hepta and hexaglutamates and decreases in penta and tetraglutamates. These results support the hypothesis that changes in polyglutamate chain length are related to the regulation of one-carbon metabolism.

Animals↗
PubMed1984

High-performance liquid chromatographic determination of the distribution of naturally occurring folic acid derivatives in rat liver.

Procedures which allow extraction and quantitation of labile, reduced folic acid derivatives in rat liver have been developed. These procedures entail extraction of hepatic folates at 100 degrees C in 2% (w/v) sodium ascorbate, 0.2 M 2-mercaptoethanol, pH 7.85. The extract was treated with conjugase to hydrolyze folate polyglutamates and reverse-phase, ion-pair high-performance liquid chromatography was used to separate the resulting monoglutamates which were measured by microbiological assay using Lactobacillus casei. Experiments with HPLC-purified standard derivatives, so treated, showed excellent stability of tetrahydropteroylglutamic acid (H4PteGlu), 10-formyl-H4PteGlu, 5-formyl-H4PteGlu, 5-methyl-H4PteGlu, and pteroylglutamic acid (PteGlu). Under these conditions, approximately 56% of H2PteGlu was recovered unchanged while about 27% was converted to PteGlu; 5,10-methylene-H4PteGlu was quantitatively recovered as H4PteGlu. These procedures were applied to the task of measuring the distribution of naturally occurring folate cofactors in rat liver. These results indicated that rat liver folates have the following compositions: 5-methyl-H4PteGlu, 37.2%; H4PteGlu, 32.7%; 10-formyl-H4PteGlu, 22.6%; and 5-formyl-H4PteGlu, 7.7%. Experiments with [3H]PteGlu injection showed that all hepatic folates had the same specific radioactivity as determined by radioassay and L. casei assay, indicating that L. casei exhibited the same growth response to all the folates detected in rat liver.

Animals↗
PubMed1982

Folic acid and its pentaglutamate leak from human erythrocyte ghosts but not from liposomes.

Liposomes were impermeable to folic acid (PteGlu) and its pentaglutamate (PteGlu5), but both folates leaked from human erythrocyte ghosts at a similar rate. PteGlu5 leaking from ghosts was recovered in suspending medium. This indicates that although neither folate penetrates phospholipid membranes, both can traverse erythrocyte membranes without chemical modification. Reduced monoglutamate folate entered erythrocyte ghosts more slowly than it exited. Leakage of folate in both directions was slower than that of urate which, unlike folate, did not exit faster than it entered human erythrocyte ghosts.

Biological Transport, Active↗
PubMed1989

Effect of polyglutamylation of 5,10-methylenetetrahydrofolate on the binding of 5-fluoro-2'-deoxyuridylate to thymidylate synthase purified from a human colon adenocarcinoma xenograft.

CH2-H4PteGlu and H4PteGlu exist in human colon adenocarcinoma xenografts predominantly in the form of polyglutamate species at concentrations of less than 3 microM. The interaction of polyglutamates of [6R]CH2-H4PteGlu in the formation and stability of [6-3H]FdUMP-thymidylate synthase-CH2-H4PteGlun ternary complexes has therefore been examined using enzyme purified from a human colon adenocarcinoma xenograft. Dissociation of these complexes was first-order and was dependent upon the concentration of folate. [6R]CH2-H4PteGlu3-6 (0.9 to 1.6 microM) were greater than 200-fold and [6R]CH2-H4PteGlu2 (18.2 microM) was 18-fold more effective than [6R]CH2-H4PteGlu1 (335 microM) at stabilizing ternary complexes for a T1/2 for dissociation of 100 min. Polyglutamylation of CH2-H4PteGlu also increased the affinity of binding of [6-3H]FdUMP to thymidylate synthase as determined by Scatchard analysis at folate concentrations of 10 microM, where the Kd in the presence of [6R]CH2-H4PteGlu1 was in the order of 4.0 x 10(-8) M, and for [6R]CH2-H4PteGlu3-5 was between 3.7 and 5.5 x 10(-9) M. To examine whether this effect was due to differences in the rates at which [6-3H]FdUMP was bound (kon) or dissociated (koff) from the enzyme, the apparent rate of [6-3H]FdUMP binding was determined in the presence of [6R]CH2H4PteGlu1, [6R]CH2-H4PteGlu3 and [6R]CH2-H4PteGlu4. The kon values were similar and were in the range of 1.7 to 2.3 x 10(6) M-1 min-1 for 10 or 20 microM folate concentrations. Differences in binding affinity determined for [6R]CH2-H4PteGlu1 and longer polyglutamate forms of [6R]CH2-H4PteGlu were thus due to differences in koff. The Vmax for the initial velocity of [6-3H]FdUMP binding was achieved at 10 microM folate. Consequently, at concentrations of CH2-H4PteGlu polyglutamates present in tumors, inhibition of thymidylate synthase by FdUMP in vivo would be expected to be transient, based upon the concentration of [6R]CH2-H4PteGlun required for maximal formation and stability of the covalent ternary complex. It would be advantageous for modulation of CH2-H4PteGlun pools to increase the concentrations of the longer polyglutamate species (n greater than or equal to 3) to maximize the interaction between FdUMP, thymidylate synthase and CH2-H4PteGlu.

Adenocarcinoma↗
PubMed1990

Changes in hepatic folylpolyglutamate pattern in phenobarbitone-treated rats.

Folate deficiency is a common unpleasant secondary effect of anticonvulsant therapy. In order to contribute to the knowledge of biochemical mechanisms leading to this condition, the effects of two i.p. high doses of phenobarbitone administered to the rat (acute treatment) on the distribution of hepatic folate derivatives have been studied. A significant decrease of unsubstituted tetrahydro- and dihydropteroylpentaglutamates and 5,10-methylenetetrahydropentaglutamates was observed. The hypothesis that a lower availability of NADPH, which is utilized for hydroxylation reactions in phenobarbitone metabolism, may limit folate reduction is proposed.

Animals↗
PubMed1991

In vitro metabolism of 5,8-dideazafolates and 5,8-dideazaisofolates by mammalian folylpoly-gamma-glutamate synthetase.

The in vitro metabolism of a variety of 5,8-dideazafolate and 5,8-dideazaisofolate analogues by pig liver folylpolyglutamate synthetase and the specificity of the enzyme for some polyglutamate derivatives of these analogues have been investigated. All 4-oxo-quinazoline analogues were metabolized to long chain polyglutamate derivatives, primarily the pentaglutamate, whereas 4-amino-quinazolines were metabolized to a lesser extent, with the accumulation of di- and triglutamate derivatives. This pattern of metabolism was consistent with the large drop in Vmax/Km and Vmax values for folylpolyglutamate synthetase observed with diglutamate derivatives of 4-aminofolate analogues. The extent of metabolism of the various analogues did not correlate with the relative substrate effectiveness of their parent monoglutamate derivatives. The 5-chloro and 5-methyl substitutions of quinazolines enhanced the addition of glutamate residues to 4-amino derivatives but markedly impaired the metabolism of 4-oxo derivatives.

Aminopterin↗
PubMed1982

Changes in the chain length of folylpolyglutamates during liver regeneration.

Partial hepatectomy significantly alters the proportions of folylpolyglutamates of different chain lengths in rat liver. Hexa- and heptaglutamates increase at the expense of tetra- and pentaglutamates. Methyl hexaglutamates account for most of the increase in hexaglutamates, and folates with formyl oxidation-level one-carbon substituents for most of the increase in heptaglutamates. These changes are reversible and believed to represent an element of regulation of one-carbon metabolism.

Animals↗