SEARCH · Search PubMed

Results for “folate”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

SourceResearch connectionFrom yearThrough year

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 91 records · Page 5Linked to original sources

PubMed1996

Effect of milk processing on the concentration of folate-binding protein (FBP), folate-binding capacity and retention of 5-methyltetrahydrofolate.

The main objective of this study was to investigate the effects of pasteurisation, UHT processing and fermentation on the concentration of folate-binding proteins (FBP) and their folate binding capacity in comparison with the retention of the most predominant folate from, 5-CH3THF. The amount of folate-binding protein (FBP) was analysed using enzyme-linked immunosorbent assay (ELISA). Unprocessed milk and pasteurised milk were found to contain similar amounts, 211 and 168 nmol/l, of FBP, respectively. UHT-processed milk and Yoghurt naturelle, both processed at temperatures above 90 degrees C, contained only 5.2 and 0.2 nmol/l FBP, respectively. As an indication of the protein-binding capacity free and protein-bound folates were analysed after charcoal treatment using the radio-protein binding assay method (RPBA). These results indicated that all folates in unprocessed milk and pasteurised milk were protein-bound, while folates in UHT-processed milk and Yoghurt naturelle occurred freely which is supported by our findings on FBP. High-performance liquid chromatography analysis indicated that unprocessed milk, pasteurised milk, UHT-processed milk and Yoghurt naturelle contained 44.8 +/- 2.1 (n = 10), 41.1 +/- 0.9 (n = 10), 36.1 +/- 1.8 (n = 10) and 35.6 +/- 9.1 micrograms/l (n = 10) 5-methyltetrahydrofolates (5-CH3THF), respectively, after deconjugation. Corresponding values for total milk folates analysed using radio-protein binding assay were 80.4 +/- 0.9 (n = 10), 64.2 +/- 2.7 (n = 10), 48.2 +/- 1.8 (n = 10) and 54.0 +/- 8.2 micrograms/l (n = 10), respectively. Hence, both methods indicated significant (P < 0.05) losses of 5-CH3THF as a result of pasteurisation, UHT processing and fermentation, compared with unprocessed milk. In spite of apparent discrepancies in folate concentrations obtained using the two different methods, these results support the equimolar ratio of FBP and folates in unprocessed and pasteurised milk when data on 5-CH3THF, obtained using HPLC were corrected for differences in recovery. Thus, heat processing of milk not only reduced the amount of 5-CH3 THF significantly, but also changed the concentration of FBP and the folate-binding capacity of FBP, which may have implications on the bioavailability of milk folates.

Animals↗

PubMed1990

Growth inhibition by homofolate in tumor cells utilizing a high-affinity folate binding protein as a means for folate internalization.

A subline (JT-1) of L1210 mouse leukemia cells that contains elevated levels of a high-affinity folate binding protein is sensitive to growth inhibition by homofolate. Inhibition was observed at nanomolar concentrations of folate or 5-formyltetrahydrofolate where the high-affinity binding protein is the predominant uptake route for folate compounds. At 1.0 nM folate, inhibition of growth by 50% occurred at 0.7 nM homofolate, and maximal inhibition exceeded 90% at homofolate concentrations above 10 nM. Homofolate also inhibited the uptake of 1.0 nM [3H]folate by L1210/JT-1 cells in 72-hr cultures, and the extent of uptake inhibition by 1.0 and 20 nM homofolate was comparable to the inhibition of cell growth by the same concentrations of homofolate. At a growth-limiting concentration of 5-formyltetrahydrofolate (0.5 nM), half-maximal inhibition of L1210/JT-1 cell growth occurred at 1.0 nM homofolate. When excess concentrations of folate (5 microM) or 5-formyltetrahydrofolate (0.5 microM) were added to the medium, no growth inhibition was observed for homofolate at concentrations up to 100 microM. Parental cells lacking the folate binding protein did not respond to homofolate either at growth-limiting (0.5 microM) or excess (5.0 microM) levels of folate. Binding measurements showed that homofolate has a high affinity for the folate-binding protein (Ki = 0.03 nM) but interacts poorly with the reduced-folate transport system (Ki = 203 microM). These results indicate that homofolate inhibits the growth of L1210 cells when intracellular folates are acquired via the high-affinity folate binding protein. The basis for this inhibition appears to be competition by homofolate for substrate binding and internalization.

Animals↗

PubMedDate not supplied

Maternal-fetal folate transfer: effect of ethanol and dietary folate deficiency.

The effects of acute ethanol treatment and dietary folate deficiency on maternal-fetal folate transfer were studied to asses the hypothesis that the potentiation of ethanol's toxic effect on the fetus during ongoing folate deficiency was due to the impairment of folate transfer. Sprague-Dawley rats were fed either a folate-deficient diet (0.2 mg/kg) or a folate-sufficient diet (2 mg/kg) for an average of 11 weeks before pregnancy and continued until gestation day 11 when they were sacrificed. On gestation day 11, pregnant rats were treated with either ethanol (2.5 g/kg body weight) or isocaloric sucrose (control) followed by an intravenous administration of 3H-folate (2 muCi/100 g body weight) at 120 minutes. At 210 minutes, maternal blood and feto-placental tissues were removed for radioactivity measurement. Folate status and ethanol treatment had no effect on the distribution of 3H-folate in maternal circulation. However, contrary to the hypothesis, the uptake of 3H-folate by feto-placental tissues was increased in folate deficiency and by ethanol treatment, indicating that folate transfer was not impaired by the ethanol treatment. Other possibilities are discussed.

Animals↗

PubMed2003

Folate intake, plasma folate and homocysteine status in a random Finnish population.

OBJECTIVE: To assess the folate status of Finnish adults using plasma folate and homocysteine as biomarkers and to evaluate dietary and supplementary folate intakes. MATERIALS AND METHODS: Plasma folate, vitamin B(12) and total homocysteine (tHcy) were determined in a random sample of 643 subjects aged 25-74 y living in the Helsinki area. The methylenetetrahydrofolate reductase (MTHFR)-genotypes were analyzed from a subsample (n=394). Dietary intake data by 24 h recall and use of vitamin supplements were collected. RESULTS: Plasma folate was normal (>/=5 nmol/l) in 99% of subjects and optimal (>/=8 nmol/l) in terms of a minimum tHcy in 90%. Mean plasma folate of non-supplement users was 13.7 and 12.9 nmol/l and tHcy 11.3 and 9.2 micro mol/l for men and women, respectively. Elevated tHcy (>14 micro mol/l) was found in 11% of subjects. Homozygote frequency for MTHFR genotype TT was 5.0% and their plasma tHcy was 14.8 micro mol/l compared to the mean of the other subjects, 10.5 micro mol/l, P<0.05. The mean dietary folate intake was 241 micro g/day (29 micro g/MJ of energy) for men and 205 micro g/day (33 micro g/MJ) for women, respectively. The main dietary sources of folate were vegetables 12%, wholemeal ryebread 11%, fruits 10%, and potato 10%. Regular supplement users (n=97) received on average 207 micro g folic acid per day from supplements. CONCLUSIONS: The folate status of Finnish adults seems to be adequate according to energy adjusted folate intake, plasma folate and homocysteine. The MTHFR homozygote frequency was low compared to other countries. Regular use of supplementary folic acid less than 300 micro g increased plasma folate, but supplemental folic acid over 300 micro g was required to lower tHcy values significantly.

Adult↗

PubMed2001

Influence of folic acid-fortified foods on folate status in a folate depletion-repletion rat model.

An increasing number of foods fortified with varying levels of folic acid are appearing in the market place, targeted either at the general population or at specific consumer groups. Although it is assumed that the folate in these products should be highly bioavailable, there is a need to carry out studies to ascertain that this is, in fact, the case. The present study investigated the ability of selected folic acid-fortified foods (targeted at different types of consumer) to increase the folate status of folate-deficient rats. Forty-two weanling male rats (Wistar strain) were fed a folate-deficient diet containing 1 % succinyl sulfathiazole (w/w) for 28 d. Following depletion, seven rats were randomly assigned to each of five repletion diets containing folic acid, Complan, Slim Fast, Opti-Fuel2 or Cola Coa calculated to provide 200 microg folate/kg of each diet. Calculations were based on folate information from the product labels. After a further 28 d, plasma, liver and kidney folate concentrations were determined by microbiological assay. Plasma homocysteine was measured by HPLC as a functional indicator of folate status. The folate content of the foods was measured by tri-enzyme extraction followed by microbiological assay. Our analyses suggest that there may be considerable inaccuracies on the part of the manufacturers in relation to the folate declarations on the product labels. Despite this, the four foods evaluated were highly effective in elevating plasma, liver and kidney folate and lowering plasma homocysteine concentrations in rats. These results lend support to the policy of food fortification with folic acid as a means of raising the folate status of the population, and in particular to the fortification of specific foods which may target areas of the population where increased folate status is most needed.

3T3 Cells↗

PubMed1991

Biological activity of racemic folate mixtures fed to folate-depleted rats.

Most commercially available tretrahydro and substituted folates are racemic mixtures of R and S isomers about pteridine-carbon 6. Some reports show the unnatural stereoisomers modify specific aspects of folate metabolism, whereas others show no significantly different biologic effects between isomers. The extent to which unnatural folate stereoisomers are inert in vivo is unclear. Possible interactions between isomers at normal dietary folate intakes were tested by comparing growth, liver and serum folate concentrations, and congener patterns of folate-depleted rats fed amino acid-based diets with 283, 566 or 1132 nmol folic acid (standards), or 566 nmol racemic 5-methyltetrahydrofolate, 5-formyltetrahydrofolate or 5,10-diformyltetrahydrofolate/kg diet for 3 wk. Growth and tissue folate levels increased with each increment in dietary folic acid. Growth and liver and serum folate concentrations of rats fed 566 nmol 5-formyltetrahydrofolate or 5-methyltetrahydrofolate/kg diet were similar to those of rats fed 283 nmol folic acid/kg diet. Rats fed 5,10-diformyltetrahydrofolate lost weight and had depressed serum folate levels, and most died. Results show that biologic activity of racemic 5-formyltetrahydrofolate and 5-methyltetrahydrofolate was half that of folic acid, and the R isomer did not affect growth at the levels fed. Addition of a second formyl group destroyed folate bioactivity. The folate-depleted rat model is useful for testing biologic effects of individual folate stereoisomers in vivo as they become commercially available.

Administration, Oral↗

PubMed1994

Homocysteine increases as folate decreases in plasma of healthy men during short-term dietary folate and methyl group restriction.

Ten healthy adult men were fed a diet low in folate and exogenous methyl groups to study the effects on folate requirement and status. The men were housed in a metabolic unit for the entire 108-d study. After a 9-d base-line period (P1), the men were fed an amino acid-defined soybean product diet for 45 d, which provided 25 micrograms/d of folate for 30 d (P2) and (with a folate supplement) 99 micrograms/d for 15 d (P3). During P2 and P3, the low methionine and choline diet was supplemented with methionine for half the subjects to vary the dietary methyl group intake. The periods were then repeated over the next 54 d (P4-P6), with a cross-over of methionine intakes in P5 and P6. Restricting dietary methyl group intake did not increase the dietary folate requirement. Plasma total homocysteine rose during folate depletion and correlated inversely with plasma folate; however, the response of homocysteine to changes in folate intake varied among individuals from very strong to absent. The results support previous suggestions that increased plasma homocysteine concentrations provide a marker of functional folate deficiency, and further indicate that individuals may differ greatly in their susceptibility to hyperhomocysteinemia due to low folate intakes. Judged by the lack of normalization of high homocysteine concentrations during folate repletion, the current folate RDA for adult men may not provide the expected margin of protection.

Adult↗

PubMedDate not supplied

Improved folate status in children and adolescents during voluntary fortification of food with folate.

OBJECTIVE: To assess longitudinal changes in folate status in South Australian children and adolescents during fortification of food with folic acid. METHODS: Sixty-nine children and adolescents (age 12.8 +/- 2.3 years), 47 with diabetes and 22 healthy controls, had their folate status assessed at the beginning of 1999 and again after a mean 1.1 +/- 0.23 years. Intake of folate at baseline was assessed with a quantitative food frequency questionnaire. RESULTS: Baseline red cell folate (mean +/- standard deviation (SD)) was 756 +/- 294.5 nmol/L and remained constant at follow up at 736 +/- 299 nmol/L (P = 0.55) in the whole group. Serum folate increased from 24.4 +/- 6.3 nmol/L to 27.2 +/- 8.8 nmol/L (P = 0.002). Children with diabetes showed a significant increase in serum folate (from 26.3 +/- 5.7-30.1 +/- 7.9, P < 0.001) and stable red cell folate (835.8 +/- 278.6 and 808.6 +/- 296.7, P = 0.51) between baseline and the second samples, while controls showed stable serum (20.4 +/- 5.7 and 21.1 +/- 7.7, P = 0.7) and red cell folate (586.6 +/- 255.9 and 579.8 +/- 240.1, P = 0.92). A third sample collected in 17 subjects after a further 9 +/- 1.3 months showed a further increase in serum and red cell folate. Mean folate intake at baseline was 301 +/- 129 micro g/day, below the mean recommended for prevention of neural tube defects. CONCLUSIONS: Voluntary fortification of food with folate is associated with improved folate status in South Australian children and adolescents, but may not be sufficient at current levels to provide maximal protection against neural tube defects at a population level.

Adolescent↗

PubMed2005

Regulation of reduced-folate transporter-1 in retinal pigment epithelial cells by folate.

PURPOSE: Reduced-folate transporter-1 (RFT-1), a typical transport protein with 12 membrane-spanning domains, transports reduced-folates, such as N5-methyltetrahydrofolate (MTF), the predominant circulating form of folate. RFT-1 is localized to the RPE apical membrane and transports folate from RPE to photoreceptor cells. We asked whether RFT-1 activity in RPE is altered under high folate conditions. MATERIALS AND METHODS: ARPE-19 cells were cultured 24, 48, or 72 h in medium containing either 0.5 nM, 5.0 nM, or 2.26 microM MTF, and the activity of RFT-1 was assessed by determining the uptake of N5-MTF. Semiquantitative reverse transcription-polymerase chain reaction and Western blot analysis were used to study RFT-1 gene and protein expression. RESULTS: Cells treated for 72 h with 2.26 microM MTF showed a significant (40%) decrease in MTF uptake compared to cells exposed to 0.5 nM or 5 nM MTF. The effect of high concentrations of folate on RFT-1 activity was specific. Kinetic analysis showed that folate-induced attenuation of RFT-1 activity was associated with a decrease in the maximal velocity of the transporter, but no change in the substrate affinity. Steady-state levels of RFT-1 mRNA and protein decreased significantly in the presence of excess folate. CONCLUSIONS: Excess folate levels downregulate RFT-1 in RPE. This study represents the first molecular analysis of the regulation of RFT-1 by folate in RPE and reveals attenuation of the activity and expression of a folate transport protein under conditions of high levels of folate.

Blotting, Western↗

PubMed1986

Transport of folate compounds in L1210 cells: kinetic evidence that folate influx proceeds via the high-affinity transport system for 5-methyltetrahydrofolate and methotrexate.

Folate is transported into L1210 mouse leukemia cells by the same system that mediates the uptake of methotrexate and reduced folate compounds. This conclusion is supported by the following observations: (a) methotrexate competitively inhibits the influx of folate and the Ki is comparable to the Kt for methotrexate influx; (b) the profile for inhibition of folate influx by methotrexate is monophasic and complete inhibition is achieved at high concentrations of the competitor; (c) folate inhibits the influx of methotrexate and the Ki is comparable to the Kt for folate influx; (d) the N-hydroxysuccinimide ester of methotrexate, a potent and specific irreversible inhibitor of the reduced folate system, also blocks the influx of folate; (e) folate and methotrexate influx are both inhibited by low concentrations of p-chloromercuriphenylsulfonate; and (f) folate influx fluctuates with the anionic composition of the medium in the same fashion as the influx of methotrexate. Measurements of folate influx can be complicated by the fact that the 3H-labeled substrate is susceptible to decomposition and that labeled breakdown products at concentrations as low as 1% contribute appreciably to the observed uptake. Of these products, 6-hydroxymethylpterin appears to account for most of the extraneous uptake. Impurities can be eliminated by subjecting the [3H]folate to preparative thin-layer chromatography immediately prior to use.

Animals↗

PubMed1976

Regulation of folate reductase synthesis in sensitive and methotrexate-resistant sarcoma 180 cells. In vitro translation and characterization of folate reductase mRNA.

A highly specific assay for folate reductase mRNA activity from Sarcoma 180 cells was developed using the rabbit reticulocyte lysate protein synthesizing system. Quantitation of in vitro folate reductase synthesis was accomplished by direct immunoprecipitation from lysate reactions. The in vitro labeled folate reductase was synthesized in a linear response to a wide range of RNA concentrations, migrated as a single prominent radioactive species upon polyacrylamide gel electrophoresis, and was indistinguishable from authentic 14C-labeled folate reductase on the basis of molecular weight and immunotitration with anti-folate reductase gamma-globulin. The assay was used to quantitate folate reductase mRNA activity in various cell lines and under several conditions known to affect folate reductase synthesis. These included (a) sensitive and methotrexate-resistant Sarcoma 180 cells, (b) two lines of resistant cells having different relative rates of folate reductase synthesis, (c) growth of methotrexate-resistant cells in the absence of methotrexate, and (d) growth phase. The results indicate that the relative rate of folate reductase synthesis in each case can be explained solely by the level of translatable folate reductase mRNA. The use of poly(U)-Sepharose and sucrose gradient fractionation procedures indicated that folate reductase mRNA contains poly(A) and has a sedimentation coefficient of approximately 14 S. These two fractionation steps were combined to achieve an approximately 90-fold purification of folate reductase mRNA over total cytoplasmic RNA.

Animals↗

PubMed1994

Effects of ageing on folate metabolism in rats fed a long-term folate deficient diet.

The present study was focused on the effects of feeding to young (2 mo. old) and aged (30 mo. old) rats either a folate repleted or a folate deficient diet for seven weeks on several markers of folate metabolism. Serum and liver folate contents were determined, and hepatic distribution of folates based on glutamic acid chain length and pteridine ring was obtained. In addition, liver s-adenosylmethionine (Ado Met) and s-adenosylhomocysteine (AdoHcy) concentrations, as well as serum homocysteine concentration have been also determined. Ageing itself seems not to be associated with lower hepatic folate content, but in serum a 50% decrease was observed. Feeding the folate deficient diet did result in significant lower (p < 0.001) liver folate content in both age groups, and in marked decreases in serum folates. These quantitative changes in the folate deficient groups were associated with an elongation of the glutamate chain length, mostly represented by a significant decrease in the proportion of pentaglutamyl derivatives, an increase of hexaglutamyl, and appearance of hepta and octaglutamyl folate derivatives. However, there were no important modifications in the pteridine ring distribution regardless of the age and level of dietary folate. In the present study, ageing seems to be associated with a significantly lower AdoMet/AdoHc ratio (65% decrease, p < 0.05), irrespective of the folic acid included in the diet.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗

PubMed1995

Distinguishing between folate receptor-alpha-mediated transport and reduced folate carrier-mediated transport in L1210 leukemia cells.

L1210 leukemia cells transport reduced folates and methotrexate via a well defined reduced folate carrier system and, in the absence of low folate selective pressure, do not express an alternate endocytotic route mediated by cell surface folate receptors. This laboratory previously described an L1210 leukemia cell line, MTXrA, with acquired resistance to methotrexate (MTX) due to the loss of mobility of the reduced folate carrier. We now report on the transfection of MTXrA with a cDNA encoding the murine homolog of the human folate receptor isoform of KB cells to produce MTXrA-TF1, which constitutively expresses high levels of FR-alpha. MTXrA-TF1 and L1210 cells were utilized to compare transport of methotrexate mediated by FR-alpha and the reduced folate carrier, respectively. Methotrexate influx in the two lines was similar when the extracellular level was 0.1 microM, but as the methotrexate concentration increased, influx via the reduced folate carrier increased in comparison to influx mediated by FR-alpha. Transport kinetics indicated both a approximately 20-fold lower influx Kb and Vmax for MTXrA-TF1 as compared to L1210 cells. The two cell lines exhibited distinct influx properties. Methotrexate influx in MTXrA-TF1 was markedly inhibited by 50 nM folic acid and metabolic poisons. In L1210 cells, 1.0 microM folic acid did not affect MTX influx, and metabolic poisons either had no effect on or increased methotrexate influx. Removal of extracellular chloride markedly inhibited transport in MTXrA-TF1 but stimulated influx in L1210 cells. When the pH was decreased to 6.2, methotrexate influx was not altered in MTXrA-TF1 but was reduced in L1210 cells. Probenecid and sulfobromophthalein inhibit methotrexate influx in both L1210 and MTXrA-TF1 cell lines; however, inhibition in MTXrA-TF1 could be accounted for on the basis of inhibition of methotrexate binding to FR-alpha. The data indicate that the reduced folate carrier and FR-alpha function independently and exhibit distinct properties. FR-alpha expressed at sufficient levels can mediate influx of MTX and folates into cells at rates comparable to the reduced folate carrier and hence has pharmacologic and physiologic importance.

Alkenes↗

PubMed2005

Maternal folate deficiency results in selective upregulation of folate receptors and heterogeneous nuclear ribonucleoprotein-E1 associated with multiple subtle aberrations in fetal tissues.

BACKGROUND: Homocysteine, which increases in folate deficiency, can upregulate folate receptors (FR) at the translational level by increasing the interaction between a short cis-element in the 5'-untranslated region of FR-alpha mRNA and heterogeneous nuclear ribonucleoprotein-E1 (hnRNP-E1). Perturbation of this RNA-protein interaction on GD8.5 induces neural tube defects and neurocristopathies in mice. FR upregulation can also reduce cell proliferation independently of folate deficiency in some human cells. Accordingly, we tested the hypothesis that sustained murine maternal folate deficiency would negatively impact pregnancy outcomes, upregulate FR, and selectively reduce fetal cell proliferation. METHODS: Dams were fed chow with various levels of folic acid added for eight weeks before and throughout pregnancy. Following sacrifice on GD17, dams were compared for folate and homocysteine status as well as pregnancy outcomes. Fetuses from some groups were evaluated by specific biochemical, molecular, and immunohistochemical studies for FR, hnRNP-E1, and apoptosis. RESULTS: When compared to dams fed a folate-replete diet, those dams on a folate-depleted diet developed reduced red cell folates and hyperhomocysteinemia and an inverse dose-dependent upregulation of FR and hnRNP-E1 on GD17 without alterations in cell number in the majority of tissues. However, FR overexpression was accompanied by a significant reduction in the net number of cells in the midgut, lung, pons, tongue, and olfactory epithelium, and with premature differentiation in dorsal root ganglion cells and dysplasia of taste buds. By contrast, in the brain, spinal cord, diaphragm, and primordium of follicles of vibrissae, there was less FR expression, which accompanied a net reduction in number of cells and architectural anomalies. Subtle "immunohistochemical footprints" of apoptosis on GD17 fetuses corresponded with net cell loss in the lung and olfactory epithelium. Upregulation of FR could be explained by a homocysteine-induced RNA-protein interaction in folate-depleted fetuses that led to a proportionate increase in murine FR biosynthesis. CONCLUSIONS: Maternal folate deficiency results in selective upregulation of FR and hnRNP-E1 associated with multiple aberrations in fetal tissues that include increased cell loss, architectural anomalies, and premature differentiation. The potential significance of these findings to explain the wide spectrum of folate-responsive birth defects in humans is discussed.

Animals↗

PubMed2002

Nitration and chlorination of folic acid by peroxynitrite and hypochlorous acid, and the selective binding of 10-nitro-folate to folate receptor beta.

The aim of this work was to characterize folates modified by ONOO(-) and HOCl and to evaluate the binding capacity of folates modified by ONOO(-) to folate receptor alpha and beta. For the modification of folate by ONOO(-), folic acid was reacted with the combination of PMA activated PMN and PAPA NONOate or chemically synthesized ONOO(-). For the modification of folate by HOCl, folic acid was reacted with the combination of MPO and H(2)O(2) or NaOCl. The structures of products were determined by 1H-NMR and MALDI-TOF mass. Nitrated folate species were identified as 10-nitro-folate and 12-nitro-folate, and chlorinated folate was identified as 12-chloro-folate. The 10-nitro-folate showed the selective binding to FR-beta, compared to folic acid.

Carrier Proteins↗

PubMed1990

Nutritional requirements of wild-type and folate transport-deficient Leishmania donovani for pterins and folates.

The nutritional requirements for folates and pterins were assessed for two strains of Leishmania donovani promastigotes, a wild-type (D1700) parental strain and a mutant derivative (MTXA5) which possesses a markedly diminished capacity to transport both [3H]folate and [3H]methotrexate (MTX). Both L. donovani strains have an absolute growth requirement for an exogenous pterin, since their proliferation could not be sustained in completely defined medium lacking either a pterin or a folate. Supplementation of the growth medium by many of a spectrum of folates and pterins could support growth of the wild-type cell line. Surprisingly, however, the MTXA5 strain could not thrive in folate-deficient medium fortified with any of the pterins that promoted wild-type cell division, including biopterin and neopterin. The relationship between the incapacity of MTXA5 cells to transport folate and methotrexate and their inability to multiply in folate-deficient medium supplemented with pterins was evaluated using genetic approaches. First, an independently generated MTX-resistant mutant, MTXB4, was isolated in 1 mM MTX. The phenotype of the MTXB4 cells was like that of MTXA5 cells since they were unable to transport [3H]MTX and [3H]folate or grow in folate-deficient medium supplemented with pterins. Second, three revertants of the MTXA5 cells were selected for their ability to grow in 1 microM biopterin. All three revertants had regained [3H]folate and [3H]MTX transport capability concomitant with growth sensitivity to methotrexate toxicity. These observations provide genetic evidence that the competence of L. donovani to transport [3H]folate and [3H]MTX and the capability of the organisms to utilize pterins as a nutritional factor are influenced by a common genetic locus.

Affinity Labels↗

PubMed1979

Folates of rat tissue. Bioassay of tissue folylpolyglutamates and a relationship of liver folypolyglutamates to nutritional folate sufficiency.

The folate content of young rat tissues extracted into boiling ascorbate was assayed by Lactobactillus casei both without and after treatment by a folate-free preparation of conjugase. The total folate content of various tissues was: liver, 8.9 microgram/g; kidney, 2.6; adrenal, 2.6; bone marrow, 2.4; spleen, 0.9; erythrocytes, 0.8; small intestinal mucosa, 0.7; small intestinal smooth muscle, 0.8; heart, 0.6; brain, 0.4, and skeletal muscle, 0.1 microgram/g tissue. For most tissues, with the exception of muscle and kidney, approximately 80% of the total folates assayed as longer chain length folylpolyglutamates. When liver folates were analyzed from rats fed folate-supplemented, control and folate-deficient diets, a relationship was found between folate nutrition and distribution of folylpolyglutamates. The proportion of total folates in the form of longer chain length folylpolyglutamates was greatest in the livers of folate-deficient rats and least in the livers of folate-supplemented rats.

Animals↗

PubMed1987

Mammalian folylpoly-gamma-glutamate synthetase. 4. In vitro and in vivo metabolism of folates and analogues and regulation of folate homeostasis.

The regulation of folate and folate analogue metabolism was studied in vitro by using purified hog liver folylpolyglutamate synthetase as a model system and in vivo in cultured mammalian cells. The types of folylpolyglutamates that accumulate in vivo in hog liver, and changes in cellular folate levels and folylpolyglutamate distributions caused by physiological and nutritional factors such as changes in growth rates and methionine, folate, and vitamin B12 status, can be mimicked in vitro by using purified enzyme. Folylpolyglutamate distributions can be explained solely in terms of the substrate specificity of folylpolyglutamate synthetase and can be modeled by using kinetic parameters obtained with purified enzyme. Low levels of folylpolyglutamate synthetase activity are normally required for the cellular metabolism of folates to retainable polyglutamate forms, and consequently folate retention and concentration, while higher levels of activity are required for the synthesis of the long chain length derivatives that are found in mammalian tissues. The synthesis of very long chain derivatives, which requires tetrahydrofolate polyglutamates as substrates, is a very slow process in vivo. The slow metabolism of 5-methyltetrahydrofolate to retainable polyglutamate forms causes the decreased tissue retention of folate in B12 deficiency. Although cellular folylpolyglutamate distributions change in response to nutritional and physiological modulations, it is unlikely that these changes play a regulatory role in one-carbon metabolism as folate distributions respond only slowly. 4-Aminofolates are metabolized to retainable forms at a slow rate compared to folates. Although folate accumulation by cells is not very responsive to changes in folylpolyglutamate synthetase levels and cellular glutamate concentrations, cellular accumulation of anti-folate agents would be highly responsive to any factor that changes the expression of folylpolyglutamate synthetase activity.

Animals↗