Comparison of folate quantification in foods by high-performance liquid chromatography-fluorescence detection to that by stable isotope dilution assays using high-performance liquid chromatography-tandem mass spectrometry

A comparison study on folate quantitation was carried out between the recently developed stable isotope dilution assay using liquid chromatography-tandem mass spectrometry (LC-MS-MS) and the frequently used HPLC with fluorimetric detection (LC-FD). By applying LC-MS-MS, spinach, wheat bread, beef, and blood plasma were found to contain 159.2, 19.8, 1.2, and 5.6 microg/100 g total folates, respectively, whereas the respective quantitative data obtained by LC-FD were 95.5, 16.2, 0.7, and 6.8 microg/100 g. In all samples, LC-MS-MS revealed superior selectivity and precision and circumvented the shortcomings of conventional LC techniques, i.e., ambiguous peak assignment as well as high detection limits for 5-formyltetrahydrofolate, 10-formylfolic acid, and folic acid. The affinity chromatography columns used in this study showed excellent cleanup performance and permitted detection limits as low as 0.1, 0.5, 0.1, 0.08, and 0.1 microg/100 g for tetrahydrofolate (H(4)folate), 5-methyl-H(4)folate, 5-formyl-H(4)folate, 10-formylfolate, and pteroylglutamic acid, respectively. Thus, a 10-fold higher sensitivity compared to solid-phase anion-exchange cartridges was achieved. However, affinity chromatography columns revealed a significantly higher affinity toward the natural vitamers than to the racemic isotopomeric standards, which has to be considered when applying the latter in stable isotope dilution assays.     

Subcellular localization of gamma-glutamyl carboxypeptidase and of folates.

The subcellular distributions of glutamyl carboxypeptidase, folate specific activities, and radioactive metabolites of injected [3H] folic acid were studied in rat liver. The specific activity of glutamyl carboxypeptidase in the lysosomal fraction was near or greater than four times that in the other subcellular fractions. The specific activity of folates was highest in the soluble fraction (102 ng folate/mg protein) and lowest in the microsomal fraction (22 ng folate/mg protein). Nuclear, mitochondrial, and lysosomal folates were 95% folate polyglutamates, and microsomal and soluble folates were 85--90% folate polyglutamates. Injected [3H] folic acid was initially concentrated in the microsomal fraction, as measured by 3h cpm per ng folate. Initially, injected [3H] folic acid was found converted to folate penta- and hexaglutamates in all fractions to a similar extent except in the microsomes where the percentage conversion was much less, as measured by the percentage of total 3H cpm determined to be [3H] folate penta- and hexaglutamates. At 24 h, the conversion of [3H] folates to penta- and hexaglutamates in each fraction was less than that found for the endogenous folates. Injected [3H] folic acid after 2h was found to consist of 94% reduced folates in the soluble fraction, 56% in the mitochondrial, 55% in the nuclear, 20% in the lysosomal, and 15% in the microsomal fraction.     

Subcellular localization of γ-glutamyl carboxypeptidase and of folates

The subcellular distributions of glutamyl carboxypeptidase, folate specific activities, and radioactive metabolites of injected [³H] folic acid were studied in rat liver. The specific activity of glutamyl carboxypeptidase in the lysosomal fraction was near or greater than four times that in the other subcellular fractions.The specific activity of folates was highest in the soluble fraction (102 ng folate/mg protein) and lowest in the microsomal fraction (22 ng folate/mg protein). Nuclear, mitochondrial, and lysosomal folates were 95% folate polyglutamates, and microsomal and soluble folates were 85–90% folate polyglutamates.Injected [³H] folic acid was initially concentrated in the microsomal fraction, as measured by ³H cpm per ng folate.Initially, injected [³H] folic acid was found converted to folate penta- and hexaglutamates in all fractions to a similar extent except in the microsomes where the percentage conversion was much less, as measured by the percentage of total ³H cpm determined to be [³H] folate penta- and hexaglutamates. At 24 h, the conversion of [³H] folates to penta- and hexaglutamates in each fraction was less than that found for the endogenous folates.Injected [³H] folic acid after 2 h was found to consist of 94% reduced folates in the soluble fraction, 56% in the mitochondrial, 55% in the nuclear, 20% in the lysosomal, and 15% in the microsomal fraction.     

Effect of high levels of dietary folic acid on folate metabolism in vitamin B12 deficiency

The effect of administering high levels of folic acid to vitamin B12-deficient animals was studied. In B12 deficiency histidine oxidation is decreased. This is the result of both decreased liver folate levels and increases in the proportion of methyltetrahydrofolates. The purpose of this study was to determine if the addition of very high levels of folic acid to B12-deficient diets could increase liver folates and thereby restore histidine oxidation. Rats were fed a soy protein B12-deficient diet containing 10% pectin which has been shown previously to accelerate B12 depletion. When this diet was supplemented with B12 and folic acid, histidine oxidation was 5.4% in 2 h and the livers contained 3.49 micrograms of folate/g. In the absence of B12, the histidine oxidation rate was 0.34% and the liver folate level was 1.33 micrograms/g. When 200 mg/kg of folic acid was added to the B12-deficient diet there was no increase in histidine oxidation (0.35%) but the liver folates were increased to 3.68 micrograms which is about the same as that with B12 supplementation. The percentage tetrahydrofolate of the total liver folates was the same with and without a high level of dietary folic acid. Thus there was an increase in the absolute level of tetrahydrofolate without any increase in folate function as measured by histidine oxidation. Red cell folate levels were the same with and without B12, which is in contrast to the markedly lower liver folate levels in B12 deficiency. These data suggest a difference between B12 regulation of folate metabolism in the liver and in the bone marrow.     

5-Methyltetrahydrofolic acid and folic acid measured in plasma with liquid chromatography tandem mass spectrometry: applications to folate absorption and metabolism

We describe a liquid chromatography (LC) tandem mass spectrometry (MS-MS) method for the determination of 5-methyltetrahydrofolic acid (5-methylTHF) and folic acid concentrations and enrichments in human plasma. It was used to study absorption and initial metabolism in five volunteers with two simultaneously administered oral test doses ([(13)C(6)]folic acid in capsules and [(2)H(2)]folic acid in a drink). [(13)C(5)]5-methylTHF and [(2)H(4)]folic acid were used as internal standards. Plasma samples (2 ml) were purified using folate binding protein affinity columns, followed by a concentration step. After LC separation, folates were detected using positive electrospray ionization MS-MS under multiple reaction monitoring conditions. Calibrations were linear for 5-methylTHF over the range 1.2 x 10(-11) (=limit of detection) to 3.2 x 10(-7)mol/L and for folic acid over the range 5 x 10(-10) (=limit of detection) to 4.5 x 10(-8)mol/L. For 5-methylTHF concentration in plasma, intraassay coefficient of variation was within 8.6% (and for unlabeled 5-methylTHF it was within 2.8%) and interassay coefficient of variation was within 9.0%. For folic acid concentrations these coefficient of variations were within 7.5% and within 6.5%, respectively. The [(13)C(6)] and [(2)H(2)] isotopomers of folic acid and 5-methylTHF were measured in the plasma of each volunteer for 8h. After accounting for the time delay due to capsule opening, the modeling results showed no significant differences in absorption time, first pass effect, and elimination rate in the folic acid test doses in capsule or drink. We conclude that LC-MS-MS offers increased sensitivity for quantification of plasma concentrations and enrichments of 5-methylTHF and folic acid and is applicable to stable-isotope studies in humans.     

Improved methods for the preparation of [3H]folate polyglutamates: biosynthesis with Lactobacillus casei and enzymatic synthesis with Escherichia coli folylpolyglutamate synthetase

Tritiated forms of polyglutamyl folates are not commercially available but are often needed for experimental uses in folate biochemistry. Thus, considerable interest exists in the preparation of polyglutamyl [³H]folates from the commercial monoglutamyl [³H]folates. However, refinement of established enzymatic and biological synthesis methods is still needed. To address this need we developed improved procedures for the conversion of monoglutamyl [³H]folates to various polyglutamyl forms. In the bacterial synthesis, Lactobacillus casei was grown in the presence of 1 ng/ml (2.27 nM) [³H]folic acid in Folic Acid Casei Medium. Washed cells were resuspended in 2% sodium ascorbate containing 10 mM β-mercaptoethanol and heated to release the folates. The extracted [³H]folates were purified on a folate-binding protein affinity column and then applied to a Sephadex G-10 column to separate the eluted poly- from the monoglutamyl folate species. High performance liquid chromatography with multichannel electrochemical detection indicated that the bacterial synthesis yielded predominantly polyglutamates of [³H]5-methyltetrahydrofolate and [³H]5-formyltetrahydrofolate (di- through heptaglutamates). The alternative method consisted of enzymatic polyglutamylation of [³H]folic acid catalyzed by recombinant Escherichia coli folylpolyglutamate synthetase. This enzymatic synthesis yielded predominantly tri-, tetra-, and pentaglutamyl species for the [³H]folate substrate.     

Effects of dietary folic acid level and symbiotic folate production on fitness and development in the fruit fly Drosophila melanogaster

Folic acid is a vitamin for probably all animals. When converted to folate forms, it is used in DNA synthesis and amino acid metabolism. Literature suggests insects must consume folates, folates do not affect others, is a toxin for some, and that a few insects synthesize it. It has been reported that Drosophila melanogaster does not consistently need dietary folate because it can synthesize it. This seems unlikely since animals generally lack this ability. More likely, folates thought to have been made by the fly came from microbial symbionts. We aimed to clarify how dietary folic acid affects fitness and development in fruit flies and whether flies may receive folates from microbial symbionts. We found larvae were more viable and developed faster with increasing dietary folic acid, with the surprising exception that larvae fed nearly-zero folic acid developed faster. Their body folate levels did not significantly differ from those that consumed up to 600 times more folic acid. However, these flies fed little folate only achieved normal body folate levels and development times when antibiotics were excluded from the diet. When flies consumed near-zero folates with antibiotics, their body folate levels decreased and development was prolonged. An assay for the endosymbiont Wolbachia in flies used to generate the experimental flies did not show presence of these bacteria. Our data suggest D. melanogaster can harbor unknown bacterial symbiont(s) that provide essential folates to their host when it is scarce in the diet, allowing the fruit fly to maintain growth and development.     

Identification of folate binding macromolecule in rabbit choroid plexus.

A macromolecular binder of folic acid and folic acid derivatives has been identified in the particulate fraction of homogenates of rabbit choroid plexus. Within the choroid plexus, there are 2.3 nmol of folate-binding activity (binder) per g of tissue. The molecular weight of the folate binder complex, separated from the particulate fraction after solubilization with Triton X-100, was 340,000 to 400,000 by Sephadex gel filtration. The partially purified binder, when freed of endogenous folates, bound equivalent amounts of both [3H]folic acid and [methyl-14C]methyltetrahydrofolic acid per mg of protein. Folic acid, homofolic acid, 5-methyltetrahydrofolic acid, and to a lesser degree, methotrexate, inhibited the binding of both [3H]folic acid and [14C]methyltetrahydrofolic acid. Binding activity, which decreased below pH = 7.0, was unaffected by pretreatment with ribonuclease but was eliminated completely by papain and a protease (Streptomyces griseus). Although dihydrofolate reductase was present in choroid plexus, the binder was distinct from dihydrofolate reductase as judged by gel filtration and methotrexate sensitivity. This high affinity binder of folates may be responsible, in part, for the rapid, saturable uptake of folic acid and methyltetrahydrofolic acid by rabbit choroid plexus in vitro.     

The metabolic fate of (2-14C)folic acid and a mixture of (2-14C)- and (3'',5'',9-3h)-folic acid in the rat.

The metabolism of [2-14C]folic acid over 13 days and a mixture of [2-14C]- and [3',5',9-3h]-folic acid in rats over a 6-day period is described. Both 14C and 3H are excreted in urine over the 6-day period, but 3H and 14C are only detectable in faeces for 2 days. A breakdown product of folic acid labelled with 3H only was found in some urine samples, but no metabolite corresponding to the part of the molecule containing 14C was detected. These experiments show that in the whole animal a substantial portion of orally administered folic acid undergoes scission shortly after administration [Blair Biochem. J. (1957) 68, 385-387] and that the retained folates are a shortage form for folate monoglutamates.     

An inverse relationship of rat liver folate polyglutamate chain length to nutritional folate sufficiency

The relative concentrations of folylpolyglutamates of differing chain length in rat liver and the uptake of exogenous [3H]folic acid (20 microCi, 20 microgram) into liver folylpolyglutamates were examined in rats maintained on (a) standard and folate-supplemented standard diets and (b) semi-defined folate-sufficient and folate-deficient diets. Folylpolyglutamates extracted from liver were cleaved to p-aminobenzoylpolyglutamates which were separated by ion-exchange chromatography. The relative concentrations and ultimate radiolabeling of longer-chain folylpolyglutamates (six, seven and eight glutamate residues) were greatest in the livers of folate-deficient rats, whereas the intermediate-chain folylpolyglutamates (three, four and five glutamate residues) were the greatest portion of total liver folates of folate-supplemented rats. Thus, the length of the polyglutamate chain added to liver folates is inversely related to the total concentration of liver folates. These data suggest that folylpolyglutamate biosynthesis in the liver may be controlled by the liver folate concentrations. In folate insufficiency such a control mechanism would serve to enhance the affinity of folates for folate-dependent enzymes and to conserve the liver folate concentration.     

Determination of 5-methyltetrahydrofolate (13C-labeled and unlabeled) in human plasma and urine by combined liquid chromatography mass spectrometry

The association of folates with the prevention of neural tube defects and reduced risk of other chronic diseases has stimulated interest in the development of techniques for the study of their bioavailability in humans. Stable isotope protocols differentiate between oral and/or intravenous test doses of folate and natural levels of folate already present in the body. An liquid chromatography/mass spectrometry (LC/MS) procedure is described that has been validated for the determination of [13C]5-methyltetrahydropteroyl monoglutamic acid ([13C]5-CH3H4PteGlu) in plasma and urine, following oral dosing of volunteers with different labeled folates. Folate binding protein affinity columns were used for sample purification prior to LC/MS determination. Chromatographic separation was achieved using a Superspher 100RP18 (4 microm) column and mobile phase of 0.1 mol/L acetic acid (pH 3.3):acetonitrile (90:10; 250 microL/min). Selected ion monitoring was conducted on the [M-H](-) ion: m/z 458 and 459 for analyzing 5-CH3H4PteGlu; m/z 464 [M+6-H](-) to determine 5-CH3H4PteGlu derived from the label dose; m/z 444 for analysis of 2H4PteGlu internal standard, and m/z 446 and 478 to confirm that there was no direct absorption of unmetabolized compounds. Calibration was linear over the range 0-9 x 10(-9) mol/L; the limits of detection and quantification were 0.2 x 10(-9) and 0.55 x 10(-9) mol/L, respectively. The mean coefficient of variation of the ratios (m/z 463/458) was 7.4%. The method has potential applications for other key folates involved in one-carbon metabolism.     

High-pressure liquid chromatography separation and determination of rat liver folates

The endogenous levels of the various folate compounds in rat liver were determined using high-pressure liquid chromatography for the rapid separation of folate monoglutamate forms with specific quantitation of the folates by microbiological analysis of eluted fractions. The eight folate derivatives that were assayable were tetrahydrofolic acid (H₄PteGlu), 5-methyl-H₄PteGlu, 10-formyl-H₄PteGlu, 5-formyl-H₄PteGlu, 5,10-methenyl-H₄PteGlu, 5,10-methylene-H₄PteGlu, H₂PteGlu, and PteGlu. New techniques for the preparation of tissues were developed in order to reduce the degradation of the folates. Tissue folates were converted to the monoglutamate form by a partially purified hog kidney polyglutamate hydrolase preparation and incubations were carried out at pH 6.0. This minimized folate degradation but still allowed for maximal polyglutamate hydrolase activity. Rapid removal of tissues was compared with freeze-clamping techniques. The major folates in rat liver were H₄PteGlu and 5-methyl-H₄PteGlu, comprising 42 and 39%, respectively, of the total liver folate pool of 27.30 nmol/g liver (about 13 μg/g liver). In addition, 10-formyl-H₄PteGlu and 5-formyl-H₄PteGlu each comprised 10% of the total folate pool. No endogenous PteGlu, H₂PteGlu, or 5,10-methylene-H₄PteGlu was detected in rat liver samples under our conditions. Distribution of ¹⁴C derived from a previous [¹⁴C]folic acid injection paralleled the distribution of folate as determined microbiologically after high-pressure liquid chromatography separation. The importance of these methods for the direct determination and estimation of flux of H₄PteGlu, 5-methyl-H₄PteGlu, and 10-formyl-H₄PteGlu in studies dealing with the folate system was emphasized.     

Determination of three different pools of reduced one-carbon-substituted folates. III. Reversed-phase high-performance liquid chromatography of the azo dye derivatives of p-aminobenzoylpoly-γ-glutamates and its application to the study of unlabeled endogenous pteroylpolyglutamates of rat liver

A new reversed-phase high-performance liquid chromatographic (hplc) method is described for the separation and quantitation of picomole amounts of the azo dye derivatives of p-aminobenzoylpoly-γ-glutamates. In conjunction with our previously described procedures for the differential cleavage of one-carbon-substituted, reduced folates, this hplc method provides a rapid, sensitive, and reproducible approach to the quantitation and chain-length determination of three pools of unlabeled endogenous pteroylpolyglutamates. Analysis of rat liver (n = 9) yielded the following results ( ): total folates 14.5 ± 1.0 nmol/g; folates of pool 1 (5,10-methylenetetrahydro- and unsubstituted tetra- and dihydrofolates) 2.65 ± 0.74 nmol/g; folates of pool 2 (5-methyltetrahydrofolates) 5.30 ± 0.36 nmol/g; and folates of pool 3 (5,10-methenyltetrahydro-, 10-formyltetrahydro-, 5-formyltetrahydro-, and 5-formiminotetrahydrofolates) 6.40 ± 1.60 nmol/g. Most of the folates of rat liver occur as penta- (7.60 ± 0.69 nmol/g) and hexaglutamates (6.00 ± 0.29 nmol/g). In pool 3 the hexaglutamates predominate. We also report experiments showing that folate patterns based on the amount of radioactive label incorporated after a pulse dose of [³H]folic acid differ at all times from the true steady-state pattern of unlabeled endogenous folates.     

Quantitation of folates and their catabolites in blood plasma, erythrocytes, and urine by stable isotope dilution assays

New stable isotope dilution assays were developed for the simultaneous quantitation of the folates 5-methyltetrahydrofolic acid, 5-formyltetrahydrofolic acid, tetrahydrofolic acid, 10-formylfolic acid, and folic acid as well as for their catabolites para-aminobenzoylglutamate (pABG) and acetyl-para-aminobenzoylglutamate (ApABG) in clinical samples. The methods were based on cleanup by strong anion exchange followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) detection. Deuterated analogues of the folates and [¹³C₅]-labeled isotopologues of the catabolites were applied as the internal standards in stable isotope dilution assays. Extraction in 4-morpholineethanesulfonic acid (MES) buffer at pH 5.0 ensured the optimum stability of folates and, in combination with solid-phase extraction (SPE) based on strong anion exchange, resulted in higher recoveries compared with other combinations of extraction buffers and SPE. The method was sensitive enough to detect pABG in plasma generally and unmetabolized folic acid in the plasma of a volunteer after oral dosage of an aqueous folic acid solution. The sum of folate catabolites increased by a factor of 2 in the urine of the latter volunteer, compared with that resulting when only water was dosed.     

Determination of tissue folate composition by affinity chromatography followed by high-pressure ion pair liquid chromatography

A recent report from this laboratory described the use of affinity chromatography for the isolation of pure folates from tissue extracts (J. Selhub, B. Darcy-Vrillon, and D. Fell (1988) Anal. Biochem. 168, 247-251). The present study was undertaken to develop chromatographic procedures for quantitative analysis of the individual folates in the affinity-purified mixture. Methods were devised whereby mixtures containing pteroylglutamates (PteGlu1-7) were batch reduced to the dihydro, H2PteGlu1-7, and tetrahydro, H4Pte-Glu1-7, forms. The 5-methylH4PteGlu1-7 and the 10-formylH4PteGlu1-7 series were prepared from H4Pte-Glu1-7. These compounds were used to calibrate a liquid chromatographic system for the resolution of folate mixtures. This system included reverse-phase ion pair chromatography and a diode array detector. A mixture containing oxidized and reduced PteGlu1-7, a total of 35 derivatives, was separated into seven clusters arranged in an order of increasing number of glutamate residues. Each cluster was represented by two or more peaks which were due to folates that differed in the pteridine ring structure but had the same number of glutamate residues. In clusters containing mono and diglutamyl derivatives the 10-formyltetrahydro-, the tetrahydro-, and the dihydrofolate forms appeared as separate peaks while those representing folic acid and 5-methyl-tetrahydrofolate derivatives eluted in coinciding peaks. This hierarchy was maintained in the following clusters except for increasing tendency of the former three forms of folates to elute in the same peak. The number of glutamate residues of any eluting folate can be determined on the basis of retention time in relation to those of the clusters. The pteridine ring structure of that same folate can be determined on the basis of its elution position within that cluster and spectral characteristics determined by the diode array detection system. If that position is common for more than one derivative then identification is based on differential spectral properties. Using uv absorption signals at 280 nm to determine indiscriminate folate activity, absorption signals at 350 nm are used to identify folic acid and dihydrofolate derivatives and signals at 258 nm are used to identify 10-formyltetrahydrofolate derivatives. These principles were incorporated into mathematic expressions which were used for quantitative resolution of simulated mixtures containing oxidized and reduced PteGlu5 and for the analysis of folate composition in rat liver, human milk, and cows milk.     

Folylpolyglutamates in Leishmania major

The intracellular folates of the protozoan parasite Leishmania major have been examined. About 95% of the exogenous [3H]folate accumulated by the protozoan is metabolized to polyglutamate conjugates within 65 hr, and the intracellular folates are about forty-fold concentrated over the folate in the medium. The predominant metabolite of folic acid is the pentaglutamate conjugate (85%), with lessor amounts of the tetraglutamate (approximately 9%) and hexaglutamate (approximately 3%), and trace (less than 2.5%) amounts of di-, tri- and hepta-glutamate conjugates. Chromatographic properties of the products indicate that the conjugates are linked through the gamma-carboxyl groups. The folylpolyglutamate distribution in Leishmania is similar to that found in mammalian tissues.     

亲和色谱和反相高效液相色谱测定菠菜和拟南芥中四氢叶酸代谢物及叶酸的含量(英文)

植物中来源于甘氨酸和丝氨酸的一碳单位转移给四氢叶酸用于四氢叶酸代谢物的生物合成.由于含量低、成份复杂以及稳定性差,植物组织中四氢叶酸代谢物和叶酸的定量分析一度是一个挑战性很强的课题.本研究旨在建立一种可靠方法测定对甲基基团要求不同的植物(例如累积甘氨酸甜菜碱的菠菜与不累积甘氨酸甜菜碱的拟南芥)中四氢叶酸代谢物和叶酸的含量,用于研究这些植物中通过叶酸途径的一碳单位通量.菠菜和拟南芥叶片在金色荧光灯下加液氮研磨,加入大鼠血浆轭合酶粗提物处理,提取物经叶酸结合蛋白琼脂糖亲和色谱柱纯化,用附有荧光和紫外检测器的高效液相色谱仪分离并测定四氢叶酸代谢物和叶酸的含量.菠菜和拟南芥叶片中单谷氨酸型N5-甲基四氢叶酸含量分别是252ng/g和64ng/g,而总N5-甲基四氢叶酸的含量分别是370ng/g和199ng/g.两种植物均检测到少量的四氢叶酸和N5-醛基四氢叶酸,但只在拟南芥叶片而非菠菜叶片中检测到叶酸.实验结果显示,菠菜中单谷氨酸型和多谷氨酸型N5-甲基四氢叶酸的含量均比拟南芥显著增多.这种样品制备和高效液相色谱方法适于测定植物中四氢叶酸代谢物和叶酸的含量.     

SLC19A3 encodes a second thiamine transporter ThTr2.

Recently, a new family of facilitative carriers has been cloned consisting of the reduced folate (SLC19A1) and the thiamine (SLC19A2) transporters. Despite a high level of sequence identity and similarity there is essentially no functional overlap between these carriers. The former transports folates and the latter thiamine. In this paper we describe the function of SLC19A3, another member of this transporter family most recently cloned, after transient transfection of the cDNA into HeLa cells. Uptake of [3H]thiamine, but not of methotrexate nor folic acid, was enhanced in SLC19A3 transfectants relative to vector control. Similarly, in the transfectants thiamine transport increased with an increase in pH with peak activity at pH approximately 7.5. While [3H]thiamine uptake was markedly inhibited by nonlabeled thiamine it was not inhibited by several organic cations in 100-fold excess. Hence this carrier has a high degree of specificity for vitamin B1. The data indicate that SLC19A3 has the characteristics of SLC19A2 (ThTr1) and represents a second thiamine transporter (ThTr2) in this family of facilitative carriers.     

Limited antioxidant effect after consumption of a single dose of tomato sauce by young males,despite a rise in plasma lycopene

This study investigated the effect of a single dose of tomato sauce on healthy male volunteers in a randomized crossover study. Healthy male subjects (n = 10) were enrolled. Placebo (rice and olive oil) or tomato (tomato sauce, rice and olive oil) meals were provided to the volunteers. Blood and urine samples were taken before consumption of meal (0 h) and 2, 4, 6, 24 and 48 h after meal. Consumption of tomato sauce increased plasma lycopene level by 5–22%, with a maximum level at 24 h (p<0.01) after the meal. Levels of plasma F₂-isoprostanes, hydroxyeicosatetraenoic acid products, allantoin and urinary 8-hydroxy-2′-deoxyguanosine did not change after either meal, but urinary F₂-isoprostanes (p<0.05) significantly decreased at 48 h compared to 0 h after the tomato sauce meal. This study showed that a single dose of tomato sauce meal had only a limited antioxidant effect in vivo.     

Folate intake and bowel cancer risk

Folate is a B vitamin required for one-carbon transfer reactions including methylation of cell macromolecules including DNA and synthesis of the purines adenosine and guanosine and the pyrimidine thymidine. Epidemiological evidence suggests that diets providing higher amounts of folates lower the risk of colo-rectal cancer (CRC) and these observations are supported by plausible biological mechanisms. Inadequate folate supply results in DNA damage through (a) the incorporation of uracil (in place of thymidine) into DNA and subsequent unsuccessful attempts at DNA repair and (b) aberrant patterns of DNA methylation. However, human intervention studies using relatively large doses (500–5,000 μg/day) of folic acid (a synthetic form of folate) have provided no evidence of benefit in terms of adenoma recurrence. Indeed, there is some evidence of potential harm in increased risk of prostate cancer. Possible reasons for the apparent divergence in findings from the observational and intervention studies include the use of (unphysiologically) large doses of folic acid in the intervention studies whereas smaller intakes of food folates appeared to offer “protection” against CRC in case–control and prospective cohort studies. With intakes of folic acid greater than 400 μg/day, unmetabolised folic acid appears in peripheral blood and there are suggestions that this folic acid may have adverse effects e.g. reduced cytotoxicity of Natural Killer cells. Until the benefit-risk relationship associated with mandatory fortification with folic acid has been clarified (and, in particular, the possible risk of inducing extra cases of bowel or other cancer), it would seem wise to delay further mandatory folic acid fortification.