Folate receptors targeted to clathrin-coated pits cannot regulate vitamin uptake.

Potocytosis is an endocytic process that is specialized for the internalization of small molecules. Recent studies on the uptake of 5-methyltetrahydrofolate by the folate receptor have suggested that the glycosyl-phosphatidylinositol anchor on this protein causes it to cluster and be internalized by caveolae instead of coated pits. To test this hypothesis directly, we have constructed a chimeric folate receptor that has the glycosyl-phosphatidylinositol anchor replaced with the transmembrane domain and cytoplasmic tail of the low density lipoprotein receptor. The cells with wild-type receptors delivered 5-methyltetrahydrofolate to the cytoplasm more rapidly than did cells expressing the chimeric receptor. This suggests that efficient delivery to the cytoplasm depends on caveolae. In sharp contrast to cells with wild-type folate receptors, cells internalizing folate by clathrin-coated pits were unable to decrease vitamin uptake when they were either folate replete or confluent.     

Hereditary dyserythropoiesis with abnormal membrane folate transport.

Dyserythropoiesis, which morphologically and serologically resembles congenital dyserythropoietic anemia type III but is not accompanied by anemia, is described in a young man. In addition to striking gigantism and multinuclearity of erythroid precursors, electron microscopy revealed widening of nuclear pores, nuclear clefts, and cytoplasmic inclusions. Membrane transport of 5-methyltetrahydrofolate by the patient's red cells was markedly reduced; total uptake, uptake velocity, and maximal velocity of uptake were all significantly less than in controls. In contrast, red cell uptake of pteroylglutamic acid was normal. Bone marrow cells in culture also showed decreased 5-methyltetrahydrofolate uptake, as well as very low thymidine incorporation. Because folate uptake by mitogen-stimulated lymphocytes was normal, the defect apparently does not involve all cell lines. These results suggest that a specific membrane defect, affecting the carrier system for reduced folate compounds, is present in this patient's erythrocytes, and perhaps, their bone marrow precursors.     

Urinary folate loss following inactivation of vitamin B12 by nitrous oxide in rats

S ummary . Rats were injected with [2-¹⁴C]H₄PteGlu daily for 3 d and thereafter one group left in air and a second group in an atmosphere of nitrous oxide/oxygen (1/1). Nitrous oxide inactivates cobalamin. The N₂O-treated rats excreted large amounts of L. casei -active folate into the urine. The urinary folate co-chromatographed with authentic ³H-labelled 5-methyltetrahydrofolate. Both groups of animals excreted ¹⁴C-labelled breakdown products in the urine but there was no evidence of increased folate catabolism in the N₂O-treated rats. It was concluded that the folate deficiency that develops in the N₂O-treated rat is due to massive urinary loss of folate. This appears to be secondary to impaired cellular uptake of folate which leads to a raised plasma folate level.     

Effect of ethanol and other aliphatic alcohols on the intestinal transport of folates

The effect of ethanol and other aliphatic alcohols on the intestinal transport of 5-methyltetrahydrofolate and folic acid was examined using everted sacs from rat jejunum. Ethanol added to the mucosal medium inhibited the transport of both folate compounds in a parallel manner, and the inhibition increased with increasing ethanol concentration (0.5-10% v/v). Ethanol at 3% v/v in the mucosal medium caused: depression in the pH dependency of the active transport of 5-methyltetrahydrofolate; higher inhibition in the transport of low concentration (0.1 microM) than high concentration (10 microM) of 5-methyltetrahydrofolate, and inhibition in the active accumulation against a concentration gradient of 5-methyltetrahydrofolate and L-leucine. Methanol, propanol and butanol also inhibited the transport of the folate compounds; and in general, the inhibitory effect increased with the increase in the number of carbon atoms in the hydrophobic chain. This study indicates that ethanol and other alcohols inhibit the intestinal transport of folates, that the degree of inhibition is related to the concentration and chain length of the alcohol, that the inhibition is not specific for folates and finally that the mechanism of inhibition is multifactorial.     

Failure of response to N5-methyltetrahydrofolate in combined folate and B12 deficiency

A patient with combined folate and B₁₂ deficiency due to tropical sprue failed to respond to the administration of N⁵-methyltetrahydrofolate when given by mouth or intravenously. After the injection of 1 μg of B₁₂ intravenously the patient became asymptomatic and laboratory tests returned to normal. Failure of response to N⁵-methyltetrahydrofolate in the presence of B₁₂ deficiency is evidence in favor of the “folate trap” hypothesis.     

Cerebral folate deficiency

Cerebral folate deficiency (CFD) is defined as any neurological syndrome associated with a low cerebrospinal fluid (CSF) concentration of 5-methyltetrahydrofolate (5MTHF) in the presence of normal peripheral folate status. CFD has a wide clinical presentation, with reported signs and symptoms generally beginning at around 4 months of age with irritability and sleep disturbances. These can be followed by psychomotor retardation, dyskinesia, cerebellar ataxia and spastic diplegia. Other signs may include deceleration of head growth, visual disturbances and sensorineural hearing loss. Identification of CFD is achieved by determining 5MTHF concentration in CSF. Once identified, CFD can in many cases be treated by administering oral folinic acid. Supplementation with folic acid is contraindicated and, if used, may exacerbate the CSF 5MTHF deficiency. Generation of autoantibodies against the folate receptor required to transport 5MTHF into CSF and mutations in the folate receptor 1 (FOLR1) gene have been reported to be causes of CFD. However, other mechanisms are probably also involved, as CFD has been reported in Aicardi-Goutiere’s and Rett syndromes and in mitochondriopathies. Several metabolic conditions and a number of widely used drugs can also lead to a decrease in the concentration of CSF 5MTHF, and these should be considered in the differential diagnosis if a low concentration of 5MTHF is found following CSF analysis.     

Receptor-mediated folate accumulation is regulated by the cellular folate content.

Cultured monkey kidney cells are shown to possess a cell-surface receptor that binds 5-methyltetrahydro[3',5',7,9-3H4]folic acid (5-methyltetrahydro[3H]folic acid) with high affinity (Kd = 3 nM at 4 degrees C). The cell-surface binding capacity for [3H]folic acid or [3H]methotrexate is equivalent to that for 5-methyltetrahydro[3H]folate, but the Kd values are 0.4 and 20 nM, respectively. These nonlabeled folates also inhibit cellular binding and accumulation of 5-methyltetrahydro[3H]folate, whereas specific moieties of folic acid (i.e., p-aminobenzoylglutamic acid, pteroic acid, and glutamic acid) had no effect. Surface-bound folate(s) can be released by incubation of the cells at pH 3.5. At 37 degrees C the bound material is slowly transferred into cells, where it becomes resistant to acid release. Within 4 hr the cells internalize about 3-4 times as much folate as is bound to the surface in the steady state. The amount of receptor activity increases markedly when cells are depleted of folate through growth in folate-depleted medium. Binding of folate was inhibited by an antibody raised against a soluble plasma folate-binding protein, suggesting that the cell-surface receptor and the circulating folate-binding protein are immunologically related. These data indicate that cells possess a high-affinity, high-specificity folate receptor whose expression is regulated by the folate content of the cell. They also suggest that a small molecule such as folate can enter cells by receptor-mediated endocytosis.     

Exchange between Parenteral and Tissue Folate in Man

Reduced folate compounds (dihydro- and tetrahydro pteroylglutamic acid) given intravenously in man exchange rapidly and completely with tissue 5-methyltetrahydrofolate which accumulates in plasma. Following an intravenous dose and before exchanging with tissue methylfolate the reduced compounds are altered to a heat stable form of folate, probably pteroylglutamic acid, in the plasma of control subjects. This does not take place in the plasma of patients with untreated pernicious anaemia where the injected folate remains in the reduced state until exchanged with tissue folate.     

Folate requirements of methotrexate-resistant human acute lymphoblastic leukemia cell lines

We studied the folate requirements of a human acute lymphoblastic leukemia cell line, MOLT-3, and methotrexate (MTX)-resistant sublines established in vitro. The requirement of pteroylglutamate (PGA) for optimal cell growth was different for each cell line. With increasing MTX resistance, there was progressive increase in PGA requirements, moving the PGA concentration-cell growth curve (dose-response curve) 1 log order of magnitude to the right. The increases in the requirement of 5-methyltetrahydrofolate (5-methyl-THF) by the resistant sublines were more pronounced than PGA requirement, moving the dose-response curve nearly 3 log orders in magnitude to the right. The concentrations in vitro of 5-methyl-THF required for optimal growth of the MTX- resistant sublines far exceeded the normal serum 5-methyl-THF concentrations known in humans. These observations show that MTX- resistant cell established in vitro in culture media containing PGA instead of 5-methyl-THF, a physiological folate, cannot be expected to grow in vivo. The collateral sensitivity of transport-impaired MTX- resistant sublines to 2,4-diamino-5-methyl-6-[(3',4',5'- trimethoxyanilino) methyl] quinazoline (trimetrexate, TMQ) was negated in the absence of PGA. With the addition of 5-methyl-THF, the parent cells became more resistant than the transport-impaired sublines to TMQ These data indicate that the collateral sensitivity of MTX resistant cells to the substituted 2,4-diaminoquinazoline is due to functional folate deficiency by virtue of the impaired transport of folate.     

New insights into carrier binding and epithelial uptake of the erythropoietic nutrients cobalamin and folate

PURPOSE OF REVIEW: In addition to malnutrition several genetic and acquired conditions may affect the homeostasis of cobalamin (vitamin B12) and folate, leading to megaloblastic anemia and other diseases. The present review describes new insight into protein handling of cobalamin and folate. RECENT FINDINGS: The recent solution of the three-dimensional structure of the cobalamin binder transcobalamin shows two separate domains enclosing the vitamin. This structure apparently also applies for the other homologous cobalamin binders, intrinsic factor and haptocorrin. Genetic studies of inherited cobalamin malabsorption and biochemical studies have now revealed that the functional receptor for uptake of intrinsic factor-vitamin cobalamin complexes also is a complex itself consisting of two different gene products, cubilin and amnionless. A role in folate uptake of megalin, an endocytic receptor for epithelial uptake of various proteins including transcobalamin, is now also indicated by the observation that megalin can mediate uptake of soluble folate receptor. SUMMARY: New data show the structure of cobalamin carriers and reveal novel proteins involved in the epithelial uptake of cobalamin and folate. Genetic abnormalities in three different genes encoding proteins in the epithelial uptake of cobalamin are now known to cause malabsorption of cobalamin and megaloblastic anemia.     

Studies of Folate Uptake by Phytohaemagglutinin-Stimulated Lymphocytes

Summary Cultures of lymphocytes with and without phytohaemagglutinin (PHA) were used as an in vitro model system to study the cellular uptake of tritiated folic acid, [³H]PteGlu and of¹⁴C labelled 5 methyltetrahydrofolic acid, 5 [methyl-¹⁴C]-H₄PteGlu. Total folate uptake by PHA-stimulated transformed lymphocytes, measured both by liquid scintillation counting and by autoradiography, was much greater than by non-stimulated mature lymphocytes. This suggests that growing and dividing cells take up folate more avidly than mature non-dividing cells. Uptake of both folate compounds, measured over a 4 hr period, was (a) approximately 80% less at 4°C than at 37°C, (b) exhibited saturation kinetics, and (c) was inhibited by methotrexate in concentrations of 10^?3 M, 10^?4 M, but was not affected by methotrexate at a concentration of 10^?6 M. The results suggest that an active transport mechanism may be at least partly involved in cellular folate uptake. The two folate compounds, PteGlu and 5 methyl H₄PteGlu, however, do not appear to share the same uptake pathway, since PteGlu did not inhibit the uptake of 5 [methyl-¹⁴C]H₄PteGlu by PHA-stimulated lymphocytes. Further, the uptake of 5 [methyl-¹⁴C]H₄PteGlu by PHA-stimulated lymphocytes from six patients with untreated pernicious anaemia was found to be significantly impaired, whereas the uptake of [³H]PteGlu by those cells was normal. Diphenylhydantoin did not show any consistent effect on folate uptake by lymphocytes. A sequential relation was found between the peak rates of RNA synthesis, folate uptake and DNA synthesis by PHA-stimulated lymphocytes. The peak rate of folate uptake occurred at 44–48 hr of culture, and preceded peak DNA synthesis by 24 hr and followed peak RNA synthesis by 24 hr. Folate uptake, however, did not appear to be directly coupled to either DNA or RNA synthesis, since actinomycin D inhibited both DNA and RNA synthesis by transformed lymphocytes over a 4 hr period without significantly affecting folate uptake.     

The heterogeneity and properties of folate binding proteins from chronic myelogenous leukemia cells

Previous studies have demonstrated that some chronic myelogenous leukemia cells contain a macromolecular binding factor for folic acid. This binder, which previously was believed to be a single factor, has now been resolved into two distinct binding proteins. Separation of each binder was obtained by DEAE chromatography of the partially purified lysate of chronic myelogenous leukemia cells. One binder has a molecular weight of 30;000-35,000, and the second binder has a molecular weight of 40,000-45,000. Both proteins bind the mono-, di-, and triglutamates of folic acid, N10-methyl-folate, dihydro-folate, and N5-methyltetrahydrofolate. Neither binder has determinants for N5- formyltetrahydrofolate or methotrexate. The preferred substrates for both binders appear to be the fully oxidized and partially reduced folates rather than the fully reduced folates. The lower-molecular- weight folate binding protein shows reversible binding with partially and fully reduced folates but irreversible binding with oxidized folates. This property suggests that this binder may have some function in the transport and storage of folate. The higher-molecular-weight folate binding protein, however, has only slight reversibility of binding with the partially and fully reduced folates, and it is therefore more difficult to postulate a physiologic function for this binding factor.     

Inhibitory effect of unconjugated bile acids on the intestinal transport of 5-methyltetrahydrofolate in rat jejunum in vitro

The effect of the unconjugated bile acids, cholic, deoxycholic, chenodeoxycholic, and ursodeoxycholic acids, and of the conjugated bile acid taurocholic acid on the mucosal-to-serosal transport and tissue uptake of the naturally occurring folate derivative, 5-methyltetrahydrofolate (5-CH3H4PteGlu) was examined in everted sacs of rat jejunum. Each of the unconjugated bile acids examined inhibited the transport and tissue uptake of 5-CH3H4PteGlu in a concentration dependent manner. At low concentrations (0.01-0.1 mM) of cholic and deoxycholic acids, no structural or functional damage to the intestinal mucosa occurred and the transport of 5-CH3H4PteGlu was inhibited competitively with Ki values of 0.114 mM and 0.055 mM for cholic and deoxycholic acids, respectively. The greater inhibition of 5-CH3H4PteGlu transport by unconjugated bile acids at 1 mM can be attributed to observed structural and functional damage to the intestinal mucosa. The addition of 2 mM lecithin to the mucosal medium failed to prevent the inhibitory effect of 0.1 mM deoxycholic acid on the transport of 0.5 microM 5-CH3H4PteGlu. Compared with the effect of unconjugated bile acids, the conjugated bile acid taurocholic acid (0.01-5 mM) showed no effect on the transport and tissue uptake of 5-CH3H4PteGlu. The results of this study show that intestinal transport and tissue uptake of 5-CH3H4PteGlu are inhibited by unconjugated bile acids in a dose-dependent fashion. The clinical and physiological implications of these observations are discussed.     

Folate biofortification in tomatoes by engineering the pteridine branch of folate synthesis

Plants are the main source of folate in human diets, but many fruits, tubers, and seeds are poor in this vitamin, and folate deficiency is a worldwide problem. Plants synthesize folate from pteridine, p-aminobenzoate (PABA), and glutamate moieties. Pteridine synthesis capacity is known to drop in ripening tomato fruit; therefore, we countered this decline by fruit-specific overexpression of GTP cyclohydrolase I, the first enzyme of pteridine synthesis. We used a synthetic gene based on mammalian GTP cyclohydrolase I, because this enzyme is predicted to escape feedback control in planta. This engineering maneuver raised fruit pteridine content by 3- to 140-fold and fruit folate content by an average of 2-fold among 12 independent transformants, relative to vector-alone controls. Most of the folate increase was contributed by 5-methyltetrahydrofolate polyglutamates and 5,10-methenyltetrahydrofolate polyglutamates, which were also major forms of folate in control fruit. The accumulated pteridines included neopterin, monapterin, and hydroxymethylpterin; their reduced forms, which are folate biosynthesis intermediates; and pteridine glycosides not previously found in plants. Engineered fruit with intermediate levels of pteridine overproduction attained the highest folate levels. PABA pools were severely depleted in engineered fruit that were high in folate, and supplying such fruit with PABA by means of the fruit stalk increased their folate content by up to 10-fold. These results demonstrate that engineering a moderate increase in pteridine production can significantly enhance the folate content in food plants and that boosting the PABA supply can produce further gains.     

Association of red blood cell 5-methyltetrahydrofolate and severity of coronary artery disease: a cross-sectional study from Shiraz, southern Iran

In this study we tested the hypothesis that red blood cell 5-methyltetrahydrofolate, a long-term marker of the folate status, is associated with the severity of coronary artery disease and whether this association is independent of homocysteine, vitamin B₁₂, plasma 5-methyltetrahydrofolate, 5,10-methyltetrahyrofolate reductase C677T genotype, and other cardiovascular risk factors. Two hundred and fifty-one angiographically documented patients aged <70 years with single, double, or triple coronary artery disease were investigated. Red blood cell 5-methyltetrahydrofolate concentrations were significantly decreased with the increasing number of diseased vessels (analysis of variance, P < 0.001). Red blood cell 5-methyltetrahydrofolate was also inversely and significantly correlated with the number of diseased vessels (r = −0.36, P < 0.001). Stepwise multiple regression analysis showed that red cell 5-methyltetrahydrofolate was a strong predictor of number of diseased vessels independent of plasma total homocysteine, 5,10-methyltetrahyrofolate reductase C677T genotype, and all other coronary artery risk factors (β = −0.002, P < 0.001, r² = 0.128). The results of this study suggest that low red blood cell 5-methyltetrahydrofolate is associated with the severity of coronary artery disease independent from plasma homocysteine and other cardiovascular risk factors.     

Synchronization of Converging Metabolic Pathways: Activation of the Cystathionine γ-Synthase of Neurospora crassa by Methyltetrahydrofolate

Methyltetrahydrofolate synchronizes the activities of the two branches of the pathway of methionine biosynthesis in Neurospora crassa by serving as an essential activator of cystathionine gamma-synthase and antagonizing the feedback inhibition of this enzyme by S-adenosylmethionine. Activation is specific for the methylated form of folate and increases with increasing glutamate content. The inability of extracts of me-1 and me-6 mutants to form cystathionine that has been previously reported is due to the absence of N(5)-methyltetrahydrofolate from these preparations. Extracts of me-1 mutants lack methyltetrahydrofolate because the organisms are deficient in methylenetetrahydrofolate reductase, and those of me-6 because their methyltetrahydrofolate is quantitatively removed by the procedure employed in the preparation of extracts. The folate of the me-6 organisms differs from that of wild type strains in consisting largely of the monoglutamate rather than higher conjugates.     

Intestinal Conversion of Folinic Acid to 5-Methyltetrahydrofolate in Man

Summary. In three adult subjects undergoing diagnostic umbilical vein catheteriza-tion, the active stereoisomer of folinic acid (dl,-5-formyltetrahydrofolate) was metabolized during absorption and appeared in hepatic portal venous plasma as 5-methyltetrahydrofolate. A small amount of 5-formyltetrahydrofolate and probably of 10-formylfolate was detected in hepatic portal venous plasma early during absorption as well.
Peripheral venous plasma folate changes in these subjects and in two achlorhydric subjects fed folinic acid were similar, suggesting that isomerization of 5-formyl- to 10-formyltetrahydrofolate by gastric acid was not necessary for its subsequent metabolism by the intestinal cell.
Similar increments in peripheral venous plasma folate followed feeding of comparable doses of folinic acid and folic acid, evidence that folinic acid may be effectively administered by mouth. When so administered, the effect in respect of all tissues except the intestine is essentially equivalent to the feeding of 5-methyltetra-hydrofolate itself.     

5-Methyltetrahydrofolate related enzymes and DNA polymerase alpha activities in bone marrow cells from patients with vitamin B12 deficient megaloblastic anemia

The activities of 5-methyltetrahydrofolate (5-CH3THF) related enzymes and DNA polymerase alpha were determined in bone marrow cells obtained from patients with vitamin B12 deficient megaloblastic anemia and compared with those from healthy volunteers and patients with hemolytic anemia. 5-CH3THF homocysteine methyltransferase activity was significantly lower than that in the control subjects. 5,10- methylenetetrahydrofolate reductase activity was only slightly elevated to that in the control subjects. DNA polymerase alpha activity was significantly higher than that in the control. High deoxyuridine suppression test values in vitamin B12 deficient bone marrow cells were improved by tetrahydrofolate, but not by 5-CH3THF. These data indicate that, even though the reverse reaction catalyzed by 5,10- methylenetetrahydrofolate reductase may be operative in vitamin B12 deficiency, it is not sufficient to correct the disturbance in folate metabolism in vitamin B12 deficiency. Increased DNA polymerase alpha activity may be due to compensation for disarranged DNA synthesis.     

Availability of monoglutamyl and polyglutamyl folates in normal subjects and in patients with coeliac sprue.

Intestinal folate absorption was assessed in six normal subjects and in four patients with coeliac sprue who were studied before and after treatment by dietary gluten exclusion. Comparisons were made of the luminal disappearance from the perfused jejunum of 3H-pteroylmonoglutamate and pteroyl 14C-glutamylhexaglutamate, and of the 48-hour urinary recovery of each isotope after perfusion and a tissue saturating dose of folic acid. The labelled urinary folates consisted of folic acid, 10-formyltetrahydrofolate, and 5-methyltetrahydrofolate. In each group urinary recovery of 3H was significantly greater than that of 14C, confirming the evidence from jejunal perfusion that the availability of monoglutamyl folate is greater than that of polyglutamyl folate. According to the urinary recovery data, both folates were poorly absorbed in untreated coeliac sprue, but were normally absorbed after treatment. Assuming uniform displacement of the absorbed labelled folates by the parenteral flushing dose, the finding of greater urinary isotope recovery than of luminal folate disappearance from the perfused proximal jejunal segment suggests an adaptation of the distal small bowel for folate absorption in coeliac sprue.     

Folate Concentration in Top,Middle and Bottom Layer of Packed Red Cells in Patients with Vitamin B12 Deficiency: Relation to Treatment

In response to specific treatment of vitamin B₁₂ deficient, anaemic patients there is an influx of folate into the young, circulating red cells. To separate new and old cells, capillary tubes filled with whole blood were centrifuged and the packed red cell column divided into top (T), middle (M) and bottom (B) layer. The newest cells are found in the T layer. The increase in red cell folate (RCF) concentration starts before, during or after the reticulocyte response, and is therefore not directly related to folate metabolism in the red precursor cells in the marrow. The low RCF concentration at the peak of the reticulocyte response in some of the cases demonstrates that the folate material, which may have been accumulated in the red precursor cells in the marrow, may be lost by the time the red cells enter the peripheral blood. The influx of folate into the young, circulating red cells is rapidly followed by an efflux of folate, suggesting that much of the folate material is still in the monoglutamate form. A new influx of folate is noted after a time lapse of from 5 to 10 d. Iron deficiency seems to prevent the uptake of folate by the circulating red cells.