Accumulation Of Folic Acid Research Articles

Functionalized Fluorescent Silica Nanoparticles for Bioimaging of Cancer Cells.

Functionalized fluorescent silica nanoparticles were designed and synthesized to selectively target cancer cells for bioimaging analysis. The synthesis method and characterization of functionalized fluorescent silica nanoparticles (50–60 nm), as well as internalization and subcellular localization in HeLa cells is reported here. The dye, rhodamine 101 (R101) was physically embedded during the sol–gel synthesis. The dye loading was optimized by varying the synthesis conditions (temperature and dye concentration added to the gel) and by the use of different organotriethoxysilanes as a second silica precursor. Additionally, R101, was also covalently bound to the functionalized external surface of the silica nanoparticles. The quantum yields of the dye-doped silica nanoparticles range from 0.25 to 0.50 and demonstrated an enhanced brightness of 230–260 fold respect to the free dye in solution. The shell of the nanoparticles was further decorated with PEG of 2000 Da and folic acid (FA) to ensure good stability in water and to enhance selectivity to cancer cells, respectively. In vitro assays with HeLa cells showed that fluorescent nanoparticles were internalized by cells accumulating exclusively into lysosomes. Quantitative analysis showed a significantly higher accumulation of FA functionalized fluorescent silica nanoparticles compared to nanoparticles without FA, proving that the former may represent good candidates for targeting cancer cells.

Novel Folate Binding Protein in Arabidopsis Expressed during Salicylic Acid-Induced Folate Accumulation.

Increasing the quantity of natural folates in plant foods is recently gaining significant interest, owing to their acute deficiencies in various populations. This study observed that foliar salicylic acid treatment enhanced the accumulation of folates in Arabidopsis, which correlated with the increase in a folate binding protein (FBP) and the expression of mRNA of a putative folate binding protein At5G27830. A protein band corresponding to ∼43 kDa was observed after resolving the affinity-purified protein on SDS-PAGE, and the partial amino acid sequence indicated that the protein is indeed At5G27830. Docking studies performed with At5G27830 confirmed specific binding of folic acid to predicted site. Heterologous expression of At5G27830 in the yeast resulted in significant uptake and accumulation of folic acid in cells. This novel study of a plant FBP will be useful for folate metabolic engineering of a wide range of crops.

Sensitive Electrochemical Determination of Folic Acid Using ex–situ Prepared Bismuth Film Electrodes

The electrochemical behavior of folic acid (FA), at the electrochemically prepared ex situ bismuth film (BiF) on glassy carbon electrode, clearly indicates electrocatalytic nature of the prepared film toward FA reduction (at –0.55 V). Scanning electron microscopy is used for morphological characterization of the prepared BiF. Accordingly, we establishing an electrochemical procedure based on square wave cathodic stripping voltammetry, preceded by accumulation of FA on the BiF electrode (BiFE). This analytical method is optimized and its analytical performance is presented. This electrode displays a two linear response range: 0.1 to 1.0 μmol L–1 and 1.0 – 10.0 μmol L–1 with sensitivity of 20.10 μA μmol–1 L and 2.28 μA μmol–1 L, respectively. Developed method was validated in compliance with spectrophotometric method. Excellent recovery and standard deviation obtained with BiFE revealed great analytical potential of the proposed method which was applied for the determination of FA in pharmaceuticals formulation.

Determination of Folic Acid at a Bismuth Film Electrode by Adsorptive Stripping Voltammetry

This article reports a new simple and sensitive method for the determination of folic acid by adsorptive stripping voltammetry. The method is based on the accumulation of folic acid at a bismuth film plated in situ on a glassy carbon substrate. In order to stabilize bismuth ions, sodium potassium tartrate was added to the supporting electrolyte. The bismuth film formation and folic acid accumulation conditions were optimized and measurements were carried without solution deaeration. The calibration graph was linear from 5 × 10−10 to 2 × 10−8 mole per liter with an accumulation time of 180 seconds with a limit of detection of 2 × 10−10 mole per liter. The relative standard deviation for 5 × 10−9 mole per liter of folic acid was 3.1 percent (n = 5). The method was successfully applied for determination of folic acid in pharmaceutical preparations.

Determination of Folic Acid by Adsorptive Stripping Voltammetry at a Lead Film Electrode

AbstractAn adsorptive stripping voltammetric procedure for the determination of folic acid at an in situ plated lead film electrode was described. Formation of lead film on a glassy carbon substrate and accumulation of folic acid was performed simultaneously from an acetate buffer solution of pH 5.6 at the potential −0.88 V. The measurements were carried out from aerated solutions. The calibration graph for an accumulation time of 300 s was linear from 2×10−9 to 5×10−8 mol L−1. The detection limit was 7×10−10 mol L−1, the relative standard deviation for 2×10−8 mol L−1 of folic acid was 3.9%. The proposed procedure was applied to folic acid determinations in pharmaceutical preparations.

Adecuación de las dosis de ácido fólico en la prevención de defectos congénitos

Synthetic folic acid (FA) is in the form of pteroylmonoglutamate (PGA), a form that does not occur in nature, where it is in form of pteroylpolyglutamate, mainly as 5-methyltetrahydrofolate. The organism transforms PGA into 5-methyltetrahydrofolate, and it has been detected that this process is saturated at doses of 0.4 mg of PGA. Recently, some concern has been expressed on the use of higher than recommended doses of FA, because of the possibility of saturating the biotransformation process and the consequent plasmatic accumulation of unmetabolized synthetic FA. The objective of this study is to analyze the doses of synthetic FA that are being currently used in Spain, as well as its secular and geographic distribution. The study was based on 16,761 mothers of non-malformed infants from the data base of the Spanish Collaborative Study of Congenital Malformations. All forms of folates (folic or folinic) have been considered under the general term of FA. Although an increasing trend in the proportion of mothers using FA in the last study period (2003-2004), only 17.37% of the mothers had FA supplements since before pregnancy. Among the rest, 71.13% started using FA once they knew they were pregnant, and 11.50% never took this vitamin. In addition, more than 70% of mothers who took FA ingested doses of 4 mg or more. The results by Spanish Autonomic Regions are quite similar, with more that 50% of the mothers (of the period 2003-2004), in the great majority of the Regions, taking mean daily doses of FA that are between 12.5 and 20-fold higher than the recommended 0.4 mg/day. Most of women are having FA after conception, or do not ingest FA at all. Most of those women who take FA use doses much higher than recommended. The results of this study show that it is necessary to spread these aspects among the health professionals implicated.

Preparation and In Vitro and In Vivo Evaluation of Technetium-99m-Labeled Folate and Methotrexate Conjugates as Tumor Imaging Agents

The cell-membrane folic acid (FA) receptors are known to be responsible for cellular accumulation of FA and FA analogs, such as methotrexate (MTX), and are overexpressed on several tumor cells. Folate, as well as antifolates (i.e., MTX), possess high affinity for the folate-receptor positive cells and tissues and were deemed useful for diagnostic imaging. We have prepared and evaluated technetium-99m (99mTc)- labeled FA and MTX analogs using MAG3 and MAG2 chelating agents in an attempt to develop folate-receptor targeting radiopharmaceuticals. Folate and MTX-conjugates after labeling with 99mTc by ligand exchange method displayed high in vitro stability in human plasma. In vitro cell binding and internalization on MCF-7 and MDA-MB-231 cells indicated the affinity and specificity of the radioconjugates toward human breast cancer cells. In mice, all radioconjugates showed rapid clearance from the blood and excretion mainly through the renal/urinary pathway, with some elimination by way of the biliary route. There was no significant accumulation of radioactivity observed in other organs, with the exception of the intestines. Uptake in the breast tumor was moderate in nude mice. These findings could be of potential diagnostic interest in designing and developing FA/MTX-based radiopharmaceuticals for tumor imaging.

Folic Acid Targeting of Protein Conjugates into Ascites Tumour Cells from Ovarian Cancer Patients

Despite a wealth of in vitro data describing the use of folic acid for drug and DNA delivery into ovarian cancer cell lines, there have been no reports describing the targeting of such compounds to freshly isolated tumour cells. We have carried out a study to determine the usefulness of folic acid as a targeting ligand for ovarian cancer by measuring the uptake of folic acid—BSA-FITC in tumour cells isolated from the ascitic fluid of ovarian cancer patients. In 7 out of 7 patients we have found folic acid mediated uptake of the fluorescently labelled albumin, with the accumulation (average cell fluorescence) and differential uptake (ratio between receptor mediated and fluid phase uptake) varying between patients. Accumulation of folic acid—albumin-FITC occurs in ascites tumour cells expressing the epithelial cell marker EMA, with a significant proportion of EMA negative cells also accumulating the conjugate. There is no correlation between cell cycle and uptake of folic acid-BSA-FITC. These results suggest that folic acid-targeting of therapeutics is a promising approach for the treatment of ovarian cancer.

Increased transport of pteridines compensates for mutations in the high affinity folate transporter and contributes to methotrexate resistance in the protozoan parasite Leishmania tarentolae.

Functional cloning led to the isolation of a novel methotrexate (MTX) resistance gene in the protozoan parasite Leishmania. The gene corresponds to orfG, an open reading frame (ORF) of the LD1/CD1 genomic locus that is frequently amplified in several Leishmania stocks. A functional ORF G-green fluorescence protein fusion was localized to the plasma membrane. Transport studies indicated that ORF G is a high affinity biopterin transporter. ORF G also transports folic acid, with a lower affinity, but does not transport the drug analog MTX. Disruption of both alleles of orfG led to a mutant strain that became hypersensitive to MTX and had no measurable biopterin transport. Leishmania tarentolae MTX-resistant cells without their high affinity folate transporters have a rearranged orfG gene and increased orfG RNA levels. Overexpression of orfG leads to increased biopterin uptake and, in folate-rich medium, to increased folate uptake. MTX-resistant cells compensate for mutations in their high affinity folate/MTX transporter by overexpressing ORF G, which increases the uptake of pterins and selectively increases the uptake of folic acid, but not MTX.

Effects of folate receptor expression following stable transfection into wild type and methotrexate transport-deficient ZR-75-1 human breast cancer cells.

Two biochemically distinct systems, the high affinity folate receptor and the lower affinity reduced-folate carrier, have each been implicated in mediating the transport of folates and antifolates into cells. Previous studies from our laboratory have shown that methotrexate accumulation into wild type (WT) ZR-75-1 human breast cancer cells involves a system with characteristics of the reduced-folate carrier, that this system is deficient in methotrexate resistant (MTXR) ZR-75-1 cells in which methotrexate transport is undetectable and that neither breast cancer cell line expresses folate receptors. In this report we examined the possible interaction of the reduced-folate carrier with folate receptors by stably transfecting both WT ZR-75-1 and MTXR ZR-75-1 cells with an expression vector containing a folate receptor cDNA. Clones of stably transfected MTXR ZR-75-1 and WT ZR-75-1 cells expressing comparable levels of folate receptors were studied and compared to the nontransfected cell lines. Although nontransfected WT and MTXR ZR-75-1 cell lines require concentrations > or = 100 nM folic acid for growth, the expression of folate receptors in transfected WT and MTXR ZR-75-1 cells permitted the growth of both cell lines in low concentrations (1 nM) of folic acid. While the defect in the reduced-folate carrier system in MTXR ZR-75-1 cells inhibits their growth in medium containing low concentrations of folinic acid (< or = 1 microM), MTXR ZR-75-1 cells expressing folate receptors display uninhibited growth in 1 nM folinic acid. The accumulation of folic acid, folinic acid, and methotrexate is enhanced in folate receptor-transfected WT ZR-75-1 cells and MTXR ZR-75-1 cells. Furthermore, the accumulation of folates and antifolate was similar in both transfected WT and MTXR ZR-75-1 cell lines that expressed folate receptors. This suggests that alterations in the reduced-folate carrier do not affect folate receptor function. We also examined the effect of folate receptor expression on the sensitivity of WT and MTXR ZR-75-1 cells to methotrexate and to the lipophillic antifolate trimetrexate. Increased folate receptor expression decreased the sensitivity of WT ZR-75-1 cells toward the antifolate trimetrexate, presumably through increased uptake of reduced folates. Although the expression of the folate receptor enhanced the growth of both cell lines in low folate concentrations, it did not affect the sensitivity of either WT or MTXR ZR-75-1 cells to methotrexate.

Mode of action of the antifertility sulphonamides: Lack of effects on folate metabolism

The effects of some antifertility sulphonamides on folate metabolism were investigated in the male rat. Subcutaneous injections of sulphanilamide at a dose of 150 mg/kg/day for 6 weeks produced a marked reduction in fertility of the treated animals. This effect was rapidly recovered by one week after drug withdrawal. Similar treatments with trimethoprim (30 mg/kg/day) or pyrimethamine (8 mg/kg/day) had virtually no effect on fertility. The synergistic effect of trimethoprim or pyrimethamine on the antifertility activity of sulphanilamide was not observed when the drugs were administered in combinations. Treatment with sulphapyridine (450 mg/kg/day for 6 weeks) failed to alter the levels of folate in the blood and the reproductive organs except the testes in which accumulation of folic acid occurred. The results suggest that the antifertility activity of sulphanilamide, sulphapyridine and perhaps some other sulphonamides is not associated with the inhibition of folate metabolism.

Folic acid transport in organ-cultured mucosa of human intestine: Evidence for distinct carriers

The transport of folic acid was investigated in organcultured endoscopic biopsy specimens of intestinal mucosa from normal subjects. In the proximal small intestine at pH 5.5 and 6.5, [3H]folic acid accumulated to concentrations 2.64- and 2.17-fold higher than those of the medium, respectively, but at pH 7.5 the concentration was same as that of the medium. Saturability of initial rates of uptake was demonstrable with respect to luminal concentrations of [H+] and folic acid. Two proton-dependent transport mechanisms were evident: (a) a carrier with a low affinity for H+ and a low capacity for folic acid, and (b) a high-affinity, high-capacity carrier (Km, 635 and 54.6 nmol/L [H+]; Vmax, 0.066 and 2.583 pmol folic acid/μL intracellular water per 10 minutes, respectively). Kinetic studies of folic acid uptake at pH 5.5 and 7.5 revealed a difference in Km for folic acid (15.76 and 34.38 μmol/L, respectively) with no change in Vmax. In the colon, folic acid did not accumulate against a concentration gradient, and the initial rate of uptake was not affected by luminal pH. The accumulation of folic acid in colonic mucosa was significantly higher at pH 5.5 than at 7.5 and showed significant regional variation, with optimal uptake in the sigmoid colon. Methotrexate inhibited folic acid uptake competitively both in the proximal small intestine and in the cecum (inhibition constant 6.9 and 54 μmol/L, respectively, at pH 5.5). These data indicate the presence of a proton-dependent, active transport of folic acid in the proximal small intestine at pH 5.0–6.5. At pH 7.5 and in the mucosa of the colon, uptake of folic acid proceeds by facilitated diffusion through a low-affinity carrier.

On the mechanism of folate transport in isolated intestinal epithelial cells.

The mechanism of folic acid (FA) uptake was studied in isolated intestinal epithelial cells prepared from 2- to 6-wk-old chicks. The cells accumulated FA, reaching a level of three- to fivefold that at equilibrium. In the presence of the metabolic inhibitors, NaN3 or KCN, FA was taken up only until equilibration while accumulation of FA was inhibited. Addition of these inhibitors at a steady state of FA accumulation caused a release of intracellular FA. The kinetics of FA uptake were found to be saturable (Km = 3.5 x 10(-5) M), indicating a carrier-mediated mechanism. The steady-state level of FA accumulation was higher as the concentration of NA+ in the medium increased from 0 to 120 mM. This stimulation of FA uptake by Na+ was not due to the stimulation of glucose uptake, because in experiments carried out in the presence of phlorizin, a glucose-transport inhibitor, FA accumulation was not diminished. It is suggested that FA is taken up by a Na+-coupled transport system.

Evolution of the phenomenon of drug-resistance. II. Studies of the extracellular and intracellular derivatives of folic acid in relation to resistance to sulphathiazole and growth factor requirements

This study is a continuation of the results published previously (Sevag &amp; Ishii 1958). It surveys quantitatively the extracellular and intracellular accumulation of p -aminobenzoic acid (PAB), pteridine, folic acid (FA) and citrovorum factor (CF) of the various sulphathiazole (ST)-sensitive and ST-resistant strains of Escherichia coli grown with and without ST. The altered enzymic activities of the resistant strain with respect to growth factor requirement is also determined. The following observations are made. The utilization of the exogenous PAB by the PAB-dependent strain is followed by the multiplication of cells. These events are followed by the extracellular accumulation of FA first and then CF. This pattern applies to other strains of E. coli and is in accordance with the well-known sequence of steps involved in the synthesis of PAB, pteridine, FA, CF and growth. It is shown that PAB accumulates principally extracellularly, and pteridine principally intracellularly. The synthesis of FA by the resistant strain is at least tenfold more resistant to ST than in the sensitive strain. In the resistant strain there is a greater intracellular than extracellular accumulation of FA and CF. In the sensitive strain this relationship is reversed. The resistant strains are inheritably capable of synthesizing greater amounts of pteridine, FA and CF. The PAB-dependent ST-sensitive strain can utilize a combination of 1-methionine and any of the purines, of 1-methionine alone, or vitamin B 12 alone in place of PAB for a partial or full growth. The related resistant strain, on the other hand, is unable to multiply in the salts-glucose medium with and without PAB. It requires a combination of 1-methionine and glycine for growth which cannot be replaced by any of the factors mentioned above. This requirement of the resistant strain for growth is analyzed as a deficiency of the enzymic transmethylations and transhydroxymethylation involving the function of CF in the resistant strain.