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PubMed2005

Targeting and trafficking of the human thiamine transporter-2 in epithelial cells.

Humans lack biochemical pathways for thiamine synthesis, so cellular requirements are met via specific carrier-mediated uptake pathways. Two proteins from the solute carrier SLC19A gene family have been identified as human thiamine transporters (hTHTRs), SLC19A1 (hTHTR1) and SLC19A2 (hTHTR2). Both of these transporters are co-expressed but are differentially targeted in polarized cell types that mediate vectorial thiamine transport (e.g. renal and intestinal epithelia). It is important to understand the domain structure of these proteins, namely which regions within the polypeptide sequence are important for physiological delivery to the cell surface, in order to understand the impact of clinically relevant mutations on thiamine transport. Here we have characterized the mechanisms regulating hTHTR2 distribution by using live cell imaging methods that resolve the targeting and trafficking dynamics of full-length hTHTR2, a series of hTHTR2 truncation mutants, as well as chimeras comprising the hTHTR1 and hTHTR2 sequence. We showed the following: (i) that the cytoplasmic COOH-tail of hTHTR2 is not essential for apical targeting in polarized cells; (ii) that delivery of hTHTR2 to the cell surface is critically dependent on the integrity of the transmembrane backbone of the polypeptide so that minimal truncations abrogate cell surface expression of hTHTR2; and (iii) video rate images of hTHTR2-containing intracellular vesicles displayed rapid bi-directional trafficking events to and from the cell surface impaired by microtubule-disrupting but not microfilament-disrupting agents as well as by overexpression of the dynactin subunit dynamitin (p50). Finally, we compared the behavior of hTHTR2 with that of hTHTR1 and the human reduced folate carrier (SLC19A1) to underscore commonalities in the cell surface targeting mechanisms of the entire SLC19A gene family.

Animals↗
PubMed1992

Molecular events in the membrane transport of methotrexate in human CCRF-CEM leukemia cell lines.

A variant line (CEM-7A) "overproducing" the reduced folate/MTX carrier system was isolated from human CCRF-CEM leukemia cells grown under selective conditions in medium containing 0.25 nM 5-formyl-THF as the sole folate source. This line exhibits a 95-fold increased Vmax for [3H]-MTX influx as compared to parental cells. The values for [3H]-MTX influx Km, efflux t1/2 and structural specificity for other (anti)folate compounds were unchanged. The amount of carrier protein, estimated by NHS-[3H]-MTX affinity labeling, was approximately 30-fold higher in CEM-7A cells than in parental cells. Influx of [3H]-MTX in CEM-7A cells was found to be down-regulated 6-7-fold after preincubation of cells with adenosine, 5-formyl-THF or 5-methyl-THF, but could be prevented exclusively by inhibitors of dihydrofolate reductase. The underlying mechanism(s) of these effects have not as yet been elucidated. A radioiodinated photoaffinity analog of MTX was used to prove the molecular events in carrier-mediated MTX uptake in parental CCRF-CEM cells, CEM-7A cells, and a line exhibiting a MTX-transport defect (CEM-MTX). Specific labeling of an 80-85 kDa membrane protein was observed in parental cells, but not in CEM/MTX cells. Uptake of photoprobe and levels of the 80-85 kDa membrane protein were significantly increased in CEM-7A cells. Due to extensive glycosylation the MW of the carrier protein in human cells seems to be substantially higher than that of its counterpart in murine L1210 leukemia cells (46-48 kDa). Pulse-labeling experiments at 37 degrees C demonstrated that in CEM-7A cells photoprobe uptake proceeds via a specific pathway. The 80-85 kDa membrane protein is involved in the initial binding and translocation of photoprobe, after which a 38 kDa cytosolic protein is responsible for further intracellular distribution. At this time, the combination of photoaffinity labeling techniques and the availability of variant cell lines overexpressing the reduced folate/MTX carrier protein has provided new insights into the MTX transport process in human leukemia cell lines. In the near future this approach should also allow a further elucidation of the regulatory aspects of carrier function.

Affinity Labels↗
PubMed1999

Isolation and characterization of genomic sequences involved in the regulation of the human reduced folate carrier gene (RFC1).

Decreased reduced folate carrier (RFC) activity has been associated with MTX resistance in experimental models of transport-mediated MTX resistance, and has been attributed to changes in the expression of RFC1, the gene that encodes a protein with this activity. RNA transcripts of RFC1 may contain any one of four distinct 5' untranslated regions (UTRs). We cloned a human genomic DNA fragment upstream from the RFC1 translation start site and determined the nucleotide sequence of a 4.8kb region that contained the exons corresponding to each of the reported UTRs. To identify regulatory elements that may be involved in RFC1 transcription, we first developed a semi-quantitative RT-PCR assay using primers specific for each of the 5' UTRs to amplify RNA transcripts containing each of the RFC1 5' exons, and found evidence for differential transcription of RFC1 non-coding exons in tissues, during development, and in MTX-resistant, transport-deficient cells. We also found that RFC1 RNA levels are cell cycle regulated and peak at the G1 to S transition. Then, using a series of RFC1 promoter-reporter fusion constructs, we identified genomic sequences that may be involved in the regulation of expression of exons 1b and 1c of the RFC1 gene. These studies therefore identify regulatory regions of RFC1 promoters and potential models of regulation in which these regions may exert control.

5' Untranslated Regions↗
PubMed1999

Characterization of the bile acid sensitive methotrexate carrier of rat liver cells.

The chemotherapeutic agent methotrexate is widely used in tumor therapy for different forms of leukemia and for the therapy of arthritis. Methotrexate is eliminated from systemic blood circulation by the liver and its transport into hepatocytes is therefore described in detail in this paper. Methotrexate uptake is energy- and sodium-dependent. The Km and the Vmax are 23 microM and 36 pmol/mg protein min, respectively. The apparent activation energy (E(app)) of methotrexate uptake (5 microM [3H]methotrexate) is 53.73 kJ/mol, which indicates an energy-dependent carrier-mediated process. Although methotrexate is a folate derivative, folate itself does not inhibit methotrexate uptake, whereas the reduced folates, dihydrofolate and tetrahydrofolate are weak uncompetitive inhibitors. In contrast, the bile acids taurocholate and cholate are effective competitive inhibitors of methotrexate uptake into hepatocytes. Further strong inhibitors are the loop diuretic bumetanide, the mycotoxin ochratoxin A and bromosulfophthalein. Because tumor patients develop drug resistance during methotrexate therapy, the uptake of methotrexate was tested in different hepatoma cell lines. In HepG2-cells and Reuber hepatoma Fao-cells the transport was non-existent or very small. However, the hepatocytoma fusion cell line HPCT-1E3, a hybrid cell line between primary rat hepatocytes and rat Reuber Fao-cells, shows an intermediate transport activity with a threefold increase of the methotrexate uptake. These results indicate the presence of a bile acid sensitive methotrexate carrier in hepatocytes which is absent in dedifferentiated hepatoma cells. The carrier differs from previously described transporters for the uptake of organic anions.

Animals↗
PubMedDate not supplied

Membrane transport of methotrexate in a squamous carcinoma cell line adapted to low folate concentrations.

Membrane transport characteristics of the folate analogue methotrexate (MTX) were studied in a human squamous carcinoma cell line of the head and neck (HNSCC) adapted to grow in tissue culture media with nanomolar reduced folate concentrations (SCC-11B-LF), as compared to SCC-11B cells grown in standard medium containing high folate concentrations. We observed that SCC-11B-LF cells exhibited a 10.5-fold increased uptake of [3H]-MTX via the reduced folate/MTX carrier system compared to SCC-11B cells. Affinity labelling of the reduced folate/MTX carrier system suggests that the up-regulation of [3H]-MTX transport mainly results from an increased rate of carrier translocation, and only to a minor extent (15-20%) from an increased amount of carrier protein. The upregulation of MTX transport resulted in a 2.4-fold increased growth inhibitory effect by MTX. These results suggest that membrane transport may play a more important role in MTX-cytotoxicity when SCC-11B cells in vitro are grown in more physiological folate concentrations.

Adaptation, Physiological↗
PubMed2007

Characteristics of reduced fat milks as influenced by the incorporation of folic acid.

Folic acid plays an important role in the prevention of neural tube defects (e.g., spina bifida and anencephaly), heart defects, facial clefts, urinary abnormalities, and limb deficiencies. Milk and milk products serve as a potential source for folic acid fortification because of the presence of folate-binding proteins that seem to be involved in folate bioavailability. Although milk is not a good source of folic acid, fortification could help in the prevention of the above-mentioned defects. The objective of this study was to examine the physicochemical characteristics of reduced fat milks fortified with folic acid. Reduced fat milks were prepared using 25, 50, 75, and 100% of the recommended dietary allowance of 400 microg of folic acid. Treatments included addition of folic acid at these levels before and after pasteurization. Color, pH, fat, protein, viscosity, folic acid concentration, folate-binding protein concentration, folate-binding protein profile, standard plate count, and coliform counts were determined on d 1, 7, 14, and 21. A consumer acceptability test was conducted on d 7. Data from the consumer panel were analyzed using ANOVA (PROC GLM) with means separation to determine the differences among treatments. Data obtained from the color, pH, fat, protein, viscosity, folic acid concentration, folate-binding protein concentration, standard plate count, and coliform counts were analyzed using the GLM with a repeated measure in time. Significant differences were determined at P < 0.05 using Tukey's Studentized Range Test. There were no differences in the electrophoretic mobility of folate-binding protein in the samples. The concentration of folic acid was significantly higher in reduced fat milks fortified with folic acid after pasteurization compared with the treatments in which folic acid was added before pasteurization. The consumer panelists did not find any significant differences in flavor, appearance, or texture of folic acid fortified reduced fat milks compared with that of the control. Fortification of reduced fat milks with folic acid can be accomplished without adversely affecting the product characteristics.

Animals↗
PubMed1998

Characterization of folate transport mediated by a low pH route in mouse L1210 leukemia cells with defective reduced folate carrier function.

Folate influx at low pH was characterized in MTXrA cells, an L1210 mouse leukemia cell line with a functional defect in the reduced folate carrier. Folic acid influx in MTXrA cells was negligible at pH 7.5, increased 13-fold as the pH was decreased to 6.0, and was indistinguishable from that in L1210 cells. In contrast, while methotrexate (MTX) influx in MTXrA cells at pH 6.0 was 15-fold higher than at pH 7.5, in L1210 cells it was decreased by half. Influx of MTX, folic acid, 5-methyltetrahydrofolate and 5-formyltetrahydrofolate in MTXrA cells was increased at pH < 7.0, but their pH optima and profile differed substantially. Influx of MTX and 5-methyltetrahydrofolate at pH 6.0 showed saturability, with a Kt of 2.65 and 0.56 microM, and a Vmax of 0.45 and 0.083 nmol/g dry wt/min, respectively. MTX influx mediated by the low pH transporter was insensitive to the anionic composition of the transport buffer and affected minimally (approximately 20%) by Na+ substitution. The anion transport inhibitors sulfobromophthalein, diisothiocyanatostilbene disulfonic acid, and acetamidoisothiocyanatostilbene disulfonic acid were not effective inhibitors of the low pH route. MTX transport at low pH did not increase in MTXrA-R16 cells, an MTXrA derivative with 10-fold overexpression of the reduced folate carrier (RFC) due to transfection with RFC1 cDNA. Inhibition of reduced folate carrier activity with acetamidoisothiocyanatostilbene disulfonic acid resulted in identical MTX influx in L1210, MTXrA, and MTXrA-R16 cells at pH 5.5. Finally, low pH-mediated MTX influx was reduced by energy inhibitors and partially inhibited by ionophores (nigericin > monensin >> valinomycin). The data indicate that L1210 and MTXrA cells express similar activities of a low pH folate transporter that has properties distinct from, and independent of, the reduced folate carrier.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗
PubMed2002

Interrelationships among conceptus size, uterine protein secretion, fetal erythropoiesis, and uterine capacity.

The interrelationships among d-11 conceptus size, d-105 placental weight, placental efficiency (the ability of the placenta to support fetal growth and development), fetal erythropoiesis, and uterine capacity were examined in 1/2 Meishan, 1/2 White crossbred gilts that were unilaterally ovariohysterectomized at 90 to 100 d of age. In Exp. 1, gilts were mated after at least one normal estrous cycle and then slaughtered at 105 d of gestation and number of fetuses and CL, placental weights, fetal weights, hematocrits, fetal plasma iron, and fetal plasma folate were measured. In Exp. 2, gilts were mated and plasma progesterone was measured on d 2 and 3 of gestation. On d 11, the length of the remaining uterine horn was recorded and the uterine horn was flushed with minimal essential medium. Number of CL, conceptus number, conceptus diameters, and total uterine flush retinol-binding protein (tRBP), acid phosphatase (tAP), and folate-binding protein (tFBP) were measured. Gilts were mated again and slaughtered at 105 d of pregnancy and the same traits measured in Group 1 were recorded. Plasma progesterone concentrations on d 2 and 3 were correlated with average conceptus diameter on d 11 (r = 0.60, P < 0.01, for each day). In contrast, tRBP (r = 0.49, P < 0.01), tAP (r = 0.53, P < 0.01), and tFBP (r = 0.51, P < 0.01) in uterine flushings on d 11 were only correlated with d-3 plasma progesterone concentrations. No correlations between d-11 average conceptus diameter or d-11 uterine length with d-105 uterine capacity were observed. Uterine capacity was negatively correlated with placental weight, fetal weight and fetal hematocrit (r = -0.36, P < 0.01; r = -0.44, P < 0.01; r = -0.32, P < 0.01; respectively). Hematocrits were correlated with fetal plasma iron (r = 0.50, P < 0.01) and folates (r = 0.44, P < 0.01). Hematocrit, plasma iron, and plasma folate were each correlated with residual fetal weights after adjusting for placental weight (a measure of placental efficiency), and accounted for 11% of the variation in this trait. These data suggest that conceptus diameter and uterine protein secretion on d 11 may be influenced by the onset of progesterone secretion by the CL, but do not support an influence of conceptus growth during early pregnancy on uterine capacity. These results also suggest that reducing placental and fetal weights will likely result in increased uterine capacity.

Acid Phosphatase↗
PubMed1992

Uptake of 5-formyltetrahydrofolate in isolated rat liver mitochondria is carrier-mediated.

We have characterized the uptake of 5-formyltetrahydrofolate into isolated rat liver mitochondria. Uptake of 5-formyltetrahydrofolate, 5-methyltetrahydrofolate and folic acid was linear for the first 2 min of incubation. 5-Methyltetrahydrofolate and 5-formyltetrahydrofolate were accumulated in mitochondria to 3.7- and 4.3-fold, respectively. 5-Formyltetrahydrofolate transport showed a pH optimum at 5.5 and was saturable with an apparent Km = 2.80 mumol.L-1 and Vmax = 3.49 pmol.min-1.mg protein-1. Uptake of 5-formyltetrahydrofolate was inhibited by the structural analog 5-methyltetrahydrofolate and, to a much lesser extent, folic acid and methotrexate. Uptake was not inhibited by various substrates of known mitochondrial transporters, e.g., succinate, malate, citrate, glutamate and phosphate, nor was uptake inhibited by the sulfhydryl reagent, N-ethylmaleimide. Uptake was not stimulated by preloading of mitochondria with malate or aspartate. Uptake of 5-formyltetrahydrofolate was not affected by the proton ionophore carbonyl cyanide 4-trifluoromethoxyphenyl-hydrazone nor by collapsing the electrical gradient with valinomycin. These results indicate that 5-formyltetrahydrofolate uptake into mitochondria via a carrier-mediated system specific for the reduced, naturally occurring folates. The folates, once in the mitochondria, may be converted to the polyglutamylated forms, which will not readily diffuse out of the mitochondria.

Animals↗
PubMed1984

Some nutritional effects of folate-binding protein in bovine milk on the bioavailability of folate to rats.

The excretions of folate compounds into both the urine and bile were investigated in rats after the administration of pteroylglutamic acid (PteGlu) with or without the folate-binding protein (FBP) prepared from bovine milk. When the sample solution, containing either free or bound [3H]PteGlu (i.e., bound to the FBP from milk), was delivered to rats intragastrically via oral intubation, the amounts of [3H]PteGlu excreted into the feces did not change. On the other hand, the urinary excretion of 3H-labeled folate compounds, especially [3H]5-methyltetrahydrofolic acid (5-CH3-H4PteGlu), after the administration of bound [3H]PteGlu was significantly lower (P less than 0.01) than that after the administration of free [3H]PteGlu. The urinary excretion of [3H]5-CH3-H4PteGlu was directly proportional to the initial amount of free [3H]PteGlu administered. The similar effect of FBP was also observed when the biliary excretion of 3H-labeled folate compounds was investigated in situ. Furthermore, the incorporation of [3H]PteGlu into folate-requiring intestinal microorganisms was considerably reduced when it was bound to FBP. These results suggest that milk FBP has some nutritional effects on the bioavailability of folate in vivo.

Animals↗
PubMed1999

Expression of folate receptor alpha in the mammalian retinol pigmented epithelium and retina.

PURPOSE: Folic acid is essential for DNA, RNA, and protein synthesis, and deficiencies in folate can lead to nutritional amblyopia and optic neuropathy. The transport of folate from the choroidal blood supply to the retina is only now beginning to be understood. The reduced-folate transporter was reported recently to be present in cultured human retinal pigment epithelial (RPE) cells and is thought to be localized to the apical region of these cells. The authors hypothesize that the RPE plays a role in the vectorial transport of folate from the choroidal blood to the neural retina and uses not only the reduced-folate transporter but also the folate receptor alpha in mediating this transport. The purpose of the present study was to determine whether the folate receptor alpha was present in the RPE and, if so, whether it was distributed along the basolateral membrane of the RPE, supporting a role for the protein in the initial steps of folate transport into the RPE. METHODS: The expression of the folate receptor alpha in mouse RPE was analyzed by reverse transcription-polymerase chain reaction (RT-PCR), functional assays, in situ hybridization, immunohistochemistry, and laser scanning confocal microscopy. RESULTS: RT-PCR analysis, cloning of the RT-PCR product, and subsequent sequencing established that folate receptor alpha mRNA transcripts are expressed in the mouse RPE/choroid and are expressed also in the neural retina. A heterologous functional expression assay using MTX(R)-ZR-75-1 cells showed that the folate receptor alpha cDNA obtained by RT-PCR from the RPE/choroid complex and the neural retina was functional as assessed by the binding of folic acid and by the uptake of N5-methyltetrahydrofolate. In situ hybridization localized the folate receptor alpha mRNA to the mouse RPE cells and to cells of the neural retina. The folate receptor alpha was detected immunohistochemically in the mouse and rat RPE and in several layers of the neural retina. Laser scanning confocal microscopy revealed the distribution of the folate receptor alpha along the basolateral region of the RPE and not the apical region. CONCLUSIONS: The present work represents the first analysis of the folate receptor alpha expression in intact mammalian retina. The receptor is present and functional in mouse RPE. It is distributed specifically along the basolateral surface of the RPE and is proposed to work in a coordinated manner with the reduced-folate transporter in the vectorial transport of folate from the choroidal blood to the neural retina.

Animals↗
PubMed1992

Lowering the cholesterol content of MA104 cells inhibits receptor-mediated transport of folate.

The folate receptor is clustered on the surface of MA104 cells in association with caveolae. This relationship is thought to be essential for the proper internalization and recycling of the receptor during the delivery of 5-methyltetrahydrofolate to the cytoplasm of folate-depleted cells. Both the clustered organization of the receptor and the integrity of caveolae are disrupted when cells are deprived of cholesterol. We now show that cholesterol depletion of MA104 cells markedly reduces the rate of 5-methyltetrahydrofolate internalization and causes a 70% decline in the number of receptors present in the internal, recycling compartment. This effect is consistent with morphologic data showing that cholesterol-depleted MA104 cells have a reduced number of caveolae as well as fewer receptors per caveolae.

Animals↗
PubMed2003

SLC19: the folate/thiamine transporter family.

The SLC19 gene family of solute carriers is a family of three transporter proteins with significant structural similarity, transporting, however, substrates with different structure and ionic charge. The three members of this gene family are expressed ubiquitously and mediate the transport of two important water-soluble vitamins, folate and thiamine. The concentrative transport of substrates mediated by the members of this gene family is energized by transcellular H(+)/OH(-) gradient. SLC19A1 is expressed at highest levels in absorptive cells where it is located in a polarized manner either in the apical or basal membrane, depending on the cell type. It mediates the transport of reduced folate and its analogs, such as methotrexate, which are anionic at physiological pH. SLC19A2 is expressed ubiquitously and mediates the transport of thiamine, a cation at physiological pH. SLC19A3 is also widely expressed and is capable of transporting thiamine. This review summarizes the current knowledge on the structural, functional, molecular and physiological aspects of the SLC19 gene family.

Animals↗
PubMed2002

Downregulation of reduced-folate transporter by glucose in cultured RPE cells and in RPE of diabetic mice.

PURPOSE: The polarized distribution of reduced-folate transporter (RFT)-1 to the apical retinal pigment epithelial (RPE) membrane was demonstrated recently. Nitric oxide (NO) significantly decreases the activity of RFT-1 in cultured RPE cells. NO is elevated in diabetes, and therefore in the present study the alteration of RFT-1 activity in RPE under conditions of high glucose was investigated. METHODS: Human ARPE-19 cells were incubated in media containing 5 mM glucose plus 40 mM mannitol (control) or 45 mM glucose for varying periods and the activity of RFT-1 was assessed by determining the uptake of [3H]-N(5)-methyltetrahydrofolate (MTF). The levels of mRNA encoding RFT-1 were determined by RT-PCR and protein levels by Western blot analysis. The activity of RFT-1 and expression of mRNA encoding RFT-1 were analyzed also in RPE of streptozotocin-induced diabetic mice. RESULTS: Exposure of RPE cells to 45 mM glucose for as short an incubation time as 6 hours resulted in a 35% decrease in MTF uptake. Kinetic analysis showed that the hyperglycemia-induced attenuation was associated with a decrease in the maximal velocity of the transporter with no significant change in the substrate affinity. Semiquantitative RT-PCR demonstrated that the mRNA encoding RFT-1 was significantly decreased in cells exposed to high glucose, and Western blot analysis showed a significant decrease in protein levels. The uptake of [3H]-MTF in RPE of diabetic mice was reduced by approximately 20%, compared with that in nondiabetic, age-matched control animals. Semiquantitative RT-PCR demonstrated that the mRNA encoding RFT-1 was decreased significantly in RPE of diabetic mice. CONCLUSIONS: These findings demonstrate for the first time that hyperglycemic conditions reduce the expression and activity of RFT-1 and may have profound implications for the transport of folate by RPE in diabetes.

Animals↗
PubMed1999

Cloning of the human thiamine transporter, a member of the folate transporter family.

We have isolated a cDNA from human placenta, which, when expressed heterologously in mammalian cells, mediates the transport of the water-soluble vitamin thiamine. The cDNA codes for a protein of 497 amino acids containing 12 putative transmembrane domains. Northern blot analysis indicates that this transporter is widely expressed in human tissues. When expressed in HeLa cells, the cDNA induces the transport of thiamine (K(t) = 2.5 +/- 0.6 microM) in a Na(+)-independent manner. The cDNA-mediated transport of thiamine is stimulated by an outwardly directed H(+) gradient. Substrate specificity assays indicate that the transporter is specific to thiamine. Even though thiamine is an organic cation, the cDNA-induced thiamine transport is not inhibited by other organic cations. Similarly, thiamine is not a substrate for the known members of mammalian organic cation transporter family. The thiamine transporter gene, located on human chromosome 1q24, consists of 6 exons and is most likely the gene defective in the metabolic disorder, thiamine-responsive megaloblastic anemia. At the level of amino acid sequence, the thiamine transporter is most closely related to the reduced-folate transporter and thus represents the second member of the folate transporter family.

Amino Acid Sequence↗
PubMed2002

Folate receptor-targeted liposomes as vectors for therapeutic agents.

The folate receptor is a cell surface protein that has recently been identified as a tumor marker, due to its differential overexpression in several malignancies. Current research indicates that folate can be covalently attached to the surface of liposomes to mediate their selective internalization by tumor cells through the folate receptor-mediated endocytic pathway. Optimized liposome formulations, characterized by improvements in drug loading, extended residence times in the circulation and improved drug release, have been developed to improve the biodistribution of therapeutic molecules. Theoretically, folate receptor-targeting can be combined with liposome encapsulation to synergistically affect disease outcome by enhancing the delivery of chemotherapeutic agents to neoplastic cells, while reducing systemic toxicities to normal tissues. The purpose of this chapter is to characterize the components of folate receptor-targeted liposomes, and summarize their applications in gene and drug delivery.

Antineoplastic Agents↗
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↗