Quantification of Cellular Folate Species by LC-MS after Stabilization by Derivatization.

Folate cofactors play a key role in one-carbon metabolism. Analysis of individual folate species is hampered by the low chemical stability and high interconvertibility of folates, which can lead to severe experimental bias. Here, we present a complete workflow that employs simultaneous extraction and stabilization of folates by derivatization. We perform reductive methylation employing stable isotope labeled reagents to retain information on the position and redox state of one-carbon units as well as the redox state of the pteridine ring. The derivatives are analyzed by a targeted LC(HILIC)-MS/MS method without the need for deconjugation, thereby also preserving the glutamation state of folates. The presented method does not only improve analyte coverage and sensitivity as compared to other published methods, it also greatly simplifies sample handling and storage. Finally, we report differences in the response of bacterial and mammalian systems to pharmacological inhibition of dihydrofolate reductase.

The contribution of minor folates to the total vitamin B9 content of Infant formula and clinical nutrition products.

In the present study an optimization of trienzyme treatment combining α-amylase, protease and γ-carboxy peptidase allowing complete sample preparation within a working day for the analysis of vitamin B9 (folate) in infant formula and adult/pediatric nutritional products is presented. The optimized sample preparation was applied to a set of samples representing most of the products in the marketplace. Results on Standard Reference Material 1849a were well in agreement with certified values. The main contributor to total folate was folic acid, 5-methyl-tetrahydrofolate was the only minor contributor in milk-based products. Soy-based formulas contained polyglutamates of 5-formyl-tetrahydrofolate. The relative contribution of polyglutamates to the total folate content remained low in the types of product included in this study. The results suggest that a simple di-enzyme treatment could be enough for these products, nevertheless, this should be carefully evaluated prior to making a decision on the use of tri- or di-enzyme treatment.

El ejercicio agudo aumenta las concentraciones de homocisteína en varones físicamente activos / Acute physical exercise increases homocysteine concentrations in young trained male subjects

Introducción: Niveles altos de Homocisteína (Hcy) se han identificado como un factor de riesgo cardiovascular. En relación con la práctica de ejercicio físico, los resultados son contradictorios. Objetivos: El objetivo del presente estudio fue determinar la influencia de ejercicios agudos máximo y submáximo sobre las concentraciones de homocisteína total (tHcy) y otros parámetros sanguíneos relacionados. Material y métodos: Diez varones (23,5 ± 1,8 años) físicamente activos realizaron una prueba incremental máxima y otra submáxima a una intensidad del 65% del consumo máximo de oxígeno (VO2max) en tapiz rodante. Se analizaron antes y después las concentraciones de tHcy, folato, vitamina B12 y creatinina séricas. Resultados: Las concentraciones de tHcy séricas aumentaron significativamente tras las pruebas de intensidad máxima (p < 0,05) y submáxima (p < 0,01). El folato y la vitamina B12 también aumentaron significativamente tras ambas pruebas (p < 0,05). Las concentraciones de creatinina aumentaron significativamente únicamente en la prueba máxima (p < 0,001). Se encontró una relación inversa entre los niveles de folato y de tHcy en todos los puntos (p < 0,05). Conclusión: Se observaron niveles altos de homocisteína después del ejercicio agudo tanto máximo como submáximo (AU)

Introduction: High levels of homocysteine (Hcy) have been identified as a cardiovascular risk factor. Regarding physical exercise, the results are contradictory. Objectives: The aim of this study was to determine the influence of maximal intensity exercise and submaximal constant exercise on total serum homocysteine concentrations (tHcy) and other related parameters. Material and methods: Ten physically active male subjects (mean age: 23.51 ± 1.84), performed two treadmill tests, a maximal test and a stable submaximal test at an intensity of 65% of maximal oxygen uptake (VO2max). Serum concentrations of tHcy, Folate, Vitamin B12 and creatinine were analysed before and after each test. Results: Significant increase in serum tHcy concentrations after the maximal (p < 0.05) and submaximal (p < 0.01) tests were observed. Folate and vitamin B12 concentrations also increased significantly after both tests (p < 0.05). Creatinine levels increased only after the maximal test (p < 0.001). A statistically significant inverse relationship was found between folate and tHcy concentrations (p < 0.05) at all the measurement points. Conclusion: THcy levels increased significantly after acute exercise in both maximum and submaximal intensity exercises (AU)

Comprehensive quantitative measurement of folate polyglutamates in human erythrocytes by ion pairing ultra-performance liquid chromatography/tandem mass spectrometry.

RATIONALE: The erythrocyte folate pool is reflective of an individual's long-term folate status; however, comprehensive quantitative determination of the various folate isoforms including polyglutamation (Glu(n)) status has posed an analytical problem. Factors complicating such analysis are the absence of authentic (isotope-labeled) standards and the large number of potential analytes. The present work presents high-throughput analytical methodology for the indirect comprehensive quantitation of the erythrocyte folate pool with commercially available standards. METHODS: The erythrocyte folate pool was determined comprehensively by utilizing a cascade of three complementary ultra-performance liquid chromatography (UPLC) tandem mass spectrometry (MS/MS) assays. In a first assay utilizing ion-pairing UPLC/MS/MS the relative (%) polyglutamation distribution (Glu(3-10)) of 5-methyltetrahydrofolate, tetrahydrofolate and 5-formyltetrahydrofolate is determined in a thermal extract obtained from packed erythrocytes, not requiring analytical standards. Quantitation of the erythrocyte folate pool was accomplished by performing two additional stable isotope dilution UPLC/MS/MS assays to determine whole blood and plasma folate content, utilizing commercially available [(13)C(5)]-labeled analogs of the Glu(1) analytes. Based on the values provided by each individual assay the comprehensive erythrocyte folate content could be calculated. RESULTS: The various assays have been validated for intra- and inter-run precision, accuracy, linearity and are robust. The method was sensitive enough to measure the comprehensive erythrocyte folate distribution in a Down's syndrome patient with extremely low folate, bearing the C677T mutation in the gene encoding for methylenetetrahydrofolate reductase. CONCLUSIONS: The erythrocyte folate pool can be comprehensively quantitated by running three complementary UPLC/MS/MS assays. The present assays are robust and allow for high-throughput analysis. The method can be utilized to support larger investigations that investigate the relationship between folate isoform and polyglutamation distribution and disease pathogenesis.

Influence of high-pressure processing on the profile of polyglutamyl 5-methyltetrahydrofolate in selected vegetables.

In plants, folate occurs predominantly as 5-methyltetrahydrofolate (5MTHF) polyglutamyl forms. Differences in stability and bioavailability of food folate compared to synthetic folic acid have been attributed to the presence of the polyglutamyl chain. High-pressure processing (HPP) was tested for whether it might shorten polyglutamyl chains of 5MTHF species in fresh vegetables by enabling action of native γ-glutamylhydrolase (GGH). A validated ultrahigh-performance reversed-phase liquid chromatography-tandem mass spectrometry method using stable isotope as internal standard was applied for characterizing 5MTHF polyglutamyl profiles. HPP conditions included 300, 450, and 600 MPa at 30 °C for 0 or 5 min, and vegetables were vacuum-packed before treatment. Investigated vegetables included cauliflower (Brassica oleracea), baby carrots (Daucus carota), and carrot greens (D. carota). HPP treatment caused conversion of polyglutamyl 5MTHF species to short-chain and monoglutamyl forms. Maximal conversion of polyglutamyl folate to monoglutamyl folate occurred at the highest pressure/time combination investigated, 600 MPa/30 °C/5 min. Under this condition, cauliflower monoglutamyl folate increased nearly 4-fold, diglutamyl folate 32-fold, and triglutamyl folate 8-fold; carrot monoglutamyl increased 23-fold and diglutamyl 32-fold; and carrot greens monoglutamyl increased 2.5-fold and the diglutamyl form 19-fold. Although some folate degradation was observed at certain intermediate HPP conditions, total 5MTHF folate was largely preserved at 600 MPa/5 min. Thus, HPP of raw vegetables is a feasible strategy for enhancing vegetable monoglutamate 5MTHF.

Quantification of key red blood cell folates from subjects with defined MTHFR 677C>T genotypes using stable isotope dilution liquid chromatography/mass spectrometry.

Red blood cell (RBC) folate levels are established at the time of erythropoiesis and therefore provide a surrogate biomarker for the average folate status of an individual over the preceding four months. Folates are present as folylpolyglutamates, highly polar molecules that cannot be secreted from the RBCs, and must be converted into their monoglutamate forms prior to analysis. This was accomplished using an individual's plasma pteroylpolyglutamate hydrolase by lysing the RBCs in whole blood at pH 5 in the presence of ascorbic acid. Quantitative conversion of formylated tetrahydrofolate derivatives into the stable 5,10-methenyltetrahydrofolate (5,10-MTHF) form was conducted at pH 1.5 in the presence of [(13)C(5)]-5-formyltetrahydrofolate. The resulting [(13)C(5)]-5,10-MTHF was then used as an internal standard for the formylated forms of tetrahydrofolate that had been converted into 5,10-MTHF as well any 5,10-MTHF that had been present in the original sample. A stable isotope dilution liquid chromatography-multiple reaction monitoring/mass spectrometry method was validated and then used for the accurate and precise quantification of RBC folic acid, 5-methyltetrahydrofolate (5-MTHF), tetrahydrofolate (THF), and 5,10-MTHF. The method was sensitive and robust and was used to assess the relationship between different methylenetetrahydrofolate reductase (MTHFR) 677C>T genotypes and RBC folate phenotypes. Four distinct RBC folate phenotypes could be identified. These were classified according to the relative amounts of individual RBC folates as type I (5-MTHF >95%; THF <5%; 5,10-MTHF <5%), type II (5-MTHF <95%; THF 5% to 20%; 5,10-MTHF <5%), type III (5-MTHF >55%; THF >20%; 5,10-MTHF >5%), and type IV (5-MTHF <55%; THF >20%; 5,10-MTHF >5%).

Concentración de vitamina B12 en suero en población puberal de la Comunidad de Madrid / Serum vitamin B12 levels in an adolescent population in Madrid

Introducción: Disponer de datos sobre la concentración de vitamina B12 en suero en niños es imprescindible para establecer unos percentiles que permitan realizar comparaciones entre regiones o países y poder plantear la suplementación de la dieta con vitaminas del grupo B como prevención secundaria frente a las enfermedades cardiovasculares. Material y métodos: Se realizó un estudio epidemiológico descriptivo de tipo transversal, con el fin de estimar las concentraciones séricas de vitamina B12 en la población escolar entre 13 y 15 años en la Comunidad de Madrid. Se realizó una determinación de folato y vitamina B12 en las muestras de sangre obtenidas en ayunas. Se determinó el genotipo C677T de la enzima metilentetrahidrofolato reductasa por reacción en cadena de la polimerasa (PCR). Resultados: Las concentraciones medias de vitamina B12 obtenidos en nuestro estudio fueron de 503 pmol/l; intervalo de confianza del 95 % (IC 95 %) (478-528 pmol/l). La mediana fue de 471 pmol/l; rango intercuartílico (337-632 pmol/l). No se encontraron diferencias estadísticamente significativas por edad o genotipo C677T. La concentración sérica de vitamina B12 fue significativamente mayor en las mujeres. La prevalencia de valores deficitarios de vitamina B12 (< 224 pmol/l) fue del 6 % en varones y del 4 % en mujeres. Conclusiones: Se presentan valores de referencia de las concentraciones de vitamina B12 sérica en población adolescente. La prevalencia de déficit de vitamina B12 es mayor en varones (AU)

Introduction: Serum vitamin B12 concentration levels in children are essential to establish values in order to compare different regions or countries, and for considering e the possibility of supplementing diets with group B vitamins as a secondary prevention against cardiovascular diseases. Material and methods: A cross-sectional epidemiological study was carried out to asses serum vitamin B12 levels in school children, 13-15 years of age, in Madrid. Folate and vitamin B12 vitamin determinations were performed on fasting blood samples. Genotype C677T of methylentetrahydrofolate reductase (MTHFR) enzyme was determined by PCR. Results: The mean vitamin B12 level obtained in our study was 503 pmol/l; CI 95 % CI (478-528 pmol/l). The median was 471 pmol/l; interquartile range (IR) (337-632 pmol/l). No statistically significant differences were found by age or C677T genotype for MTHFR. Serum vitamin B12 concentrations were significantly higher in females. Prevalence of vitamin B12 deficiency (< 224 pmol/l) was 6 % in males and 4 % in females. Conclusions: Reference values for serum vitamin B12 concentrations in an adolescent population are presented. Prevalence of vitamin B12 deficiencies is higher in males (AU)

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 [(3)H]folates from the commercial monoglutamyl [(3)H]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 [(3)H]folates to various polyglutamyl forms. In the bacterial synthesis, Lactobacillus casei was grown in the presence of 1 ng/ml (2.27 nM) [(3)H]folic acid in Folic Acid Casei Medium. Washed cells were resuspended in 2% sodium ascorbate containing 10mM beta-mercaptoethanol and heated to release the folates. The extracted [(3)H]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 [(3)H]5-methyltetrahydrofolate and [(3)H]5-formyltetrahydrofolate (di- through heptaglutamates). The alternative method consisted of enzymatic polyglutamylation of [(3)H]folic acid catalyzed by recombinant Escherichia coli folylpolyglutamate synthetase. This enzymatic synthesis yielded predominantly tri-, tetra-, and pentaglutamyl species for the [(3)H]folate substrate.

Automated generation of a dihydropyrimidine compound library using microwave-assisted processing.

Here we report the generation of a small focused library of 12 diversely functionalized dihydropyrimidine (DHPM) derivatives via one-pot three-component Biginelli cyclocondensation of beta-ketoesters, aldehydes and (thio)ureas. By applying controlled microwave heating under sealed vessel conditions using a fully automated microwave instrument including a gripper and liquid handler, the sequential synthesis of DHPMs can be performed in a shorter reaction time (10-20 min per one DHPM derivative) compared to conventional heating methods, which normally require several hours of reflux heating. The solid products either crystallize directly upon cooling or can be precipitated upon addition of water, requiring only filtration for isolation. In this way, the DHPM derivatives are obtained in high purity and no further purification by recrystallization or chromatography is necessary. This can be ascribed to the microwave heating technology where less side-product formation is often seen. The preparation of this 12-membered DHPM library can be carried out within approximately 9 h.

Prostate specific membrane antigen (PSMA) expression gives prostate cancer cells a growth advantage in a physiologically relevant folate environment in vitro.

BACKGROUND: Prostate specific membrane antigen (PSMA) expression is correlated with stage and grade of prostate cancer suggesting that it confers a growth advantage. We studied if PSMA folate hydrolase activity provides cells a growth advantage in a low folate (LF) micro-environment by hydrolyzing extracellular poly-gamma-glutamated folate to a form that cells can import. METHODS: Proliferation of LNCaP and DU-145 cells was assessed in media containing low (LF), physiological (PF), or high (HF) folate with or without penta-gamma-glutamated folate and a PSMA specific folate hydrolase inhibitor, 2-(phosphonomethyl)-pentanedioic acid (2-PMPA). RESULTS: LNCaP cells, which express PSMA, and DU-145 cells, which do not, displayed decreased proliferation when grown in LF or PF compared to HF media. This reduction in proliferation was eliminated in LNCaP cells when penta-gamma-glutamated folate was added to the media. In the presence of penta-gamma-glutamated folic acid DU-145 cells displayed increased growth but this was still significantly lower than growth in HF medium. Addition of 2-PMPA attenuated the increased growth seen in LNCaP cells but had no effect on DU-145 cell growth. CONCLUSIONS: The folate hydrolase activity of PSMA may provide a growth advantage in LF and PF environments.

Capillary electrophoresis and high-performance liquid chromatography determination of polyglutamyl 5-methyltetrahydrofolate forms in citrus products.

The 5-methyltetrahydrofolate (5mTHF) polyglutamates in citrus products were analyzed by capillary electrophoresis (CE) and high-performance liquid chromatography (HPLC). Folate species were purified from citrus products and concentrated from 2- to 100-fold using combined folate-affinity chromatography and C18 extraction. Seven polyglutamyl 5mTHFs were found in most not-from-concentrate (NFC) orange juices (OJ) in total amounts of approximately 1 nmol/mL, with varying distributions of individual polyglutamates. Folate amounts and distributions were also measured in orange fractions, single-strength OJ from concentrate, NFC grapefruit juice, and citrus peel molasses. Models containing ascorbic acid had folate thermal degradation rates one-seventh that of models without ascorbic acid. Pasteurization studies demonstrated that folate loss was <2% for commercial OJ pasteurization conditions (i.e., 93 degrees C for 5 s, 88 degrees C for 15 s, and 82 degrees C for 30 s). Both methods were precise, reproducible, and potentially faster than traditional analytical procedures requiring enzymatic deconjugation and microbial assays.

Effects of cultivation conditions on folate production by lactic acid bacteria.

A variety of lactic acid bacteria were screened for their ability to produce folate intracellularly and/or extracellularly. Lactococcus lactis, Streptococcus thermophilus, and Leuconostoc spp. all produced folate, while most Lactobacillus spp., with the exception of Lactobacillus plantarum, were not able to produce folate. Folate production was further investigated in L. lactis as a model organism for metabolic engineering and in S. thermophilus for direct translation to (dairy) applications. For both these two lactic acid bacteria, an inverse relationship was observed between growth rate and folate production. When cultures were grown at inhibitory concentrations of antibiotics or salt or when the bacteria were subjected to low growth rates in chemostat cultures, folate levels in the cultures were increased relative to cell mass and (lactic) acid production. S. thermophilus excreted more folate than L. lactis, presumably as a result of differences in the number of glutamyl residues of the folate produced. In S. thermophilus 5,10-methenyl and 5-formyl tetrahydrofolate were detected as the major folate derivatives, both containing three glutamyl residues, while in L. lactis 5,10-methenyl and 10-formyl tetrahydrofolate were found, both with either four, five, or six glutamyl residues. Excretion of folate was stimulated at lower pH in S. thermophilus, but pH had no effect on folate excretion by L. lactis. Finally, several environmental parameters that influence folate production in these lactic acid bacteria were observed; high external pH increased folate production and the addition of p-aminobenzoic acid stimulated folate production, while high tyrosine concentrations led to decreased folate biosynthesis.

Influence of processing on total, monoglutamate and polyglutamate folate contents of leeks, cauliflower, and green beans.

Bioavailability of dietary folate might be impaired by the polyglutamate chain to which approximately 70% of dietary folates are bound. This chain must be removed enzymatically in the intestine before folate is absorbed as a monoglutamate. To increase formation of monoglutamate folate in vegetables, the vegetables were subjected to various processing treatments. Treatments included freezing (-18 degrees C, 16 h) and thawing (4 degrees C, 24 h) and hydrostatic high-pressure treatment (200 MPa, 5 min). Both freezing/thawing and high-pressure treatment increased the proportion of folate in the monoglutamate form in leeks, cauliflower, and green beans 2-3-fold. However, loss of total folate after these treatments was >55%. It is concluded that conversion of folate polyglutamate to the monoglutamate form in vegetables is possible by certain processing treatments. Potentially this could lead to vegetables with higher folate bioavailability. However, to prevent folate loss into processing water, processing in a closed system should be applied.

Folylpolyglutamate synthetase in Lactobacillus leichmannii: in vitro synthesis and characterization of polyglutamylfolates.

In vitro synthesis of folylpolyglutamates by folylpolyglutamate synthetase from Lactobacillus leichmannii has been studied and optimal conditions for enzyme activity determined. It is found that while ATP (5 mM) is essential for the synthesis of folylpolyglutamates homocysteine augments the same. Replacement of vitamin B12 (2 ng/ml) with deoxyuridine (20 micrograms/ml) in growth medium does not alter the enzymatic parameters studied. DEAE-cellulose column chromatography of in vitro synthesised folylpolyglutamates indicates that folylpolyglutamate synthetase of L. leichmannii can synthesize polyglutamates up to a chain length of four glutamate residues.

Inability of leucovorin to rescue a naturally methotrexate-resistant human soft tissue sarcoma cell line from trimetrexate cytotoxicity.

A human lymphoblastoid line (RPMI-1788), a methotrexate-sensitive human fibrosarcoma cell line (HT-1080), and a naturally resistant mixed mesodermal human sarcoma cell line with impaired methotrexate polyglutamylation (HS-42), recently established in our laboratory, were used to compare the ability of leucovorin to prevent trimetrexate cytotoxicity. Growth inhibition and an in situ thymidylate synthesis activity assay showed that inhibitory effects of trimetrexate (1 to 10 microM), 24-h exposure, were prevented by 10 microM leucovorin in the RPMI-1788 and HT-1080 cell lines but not in the HS-42 cell line. Total intracellular reduced folates increased about 2-fold in the three cell lines after exposure to leucovorin (10 microM) for 4 h, and after a 6-hour efflux remained elevated (1.5- and 1.3-fold of control levels) in RPMI-1788 and HT-1080 cells but decreased to 80% of control levels in HS-42 cells. Although uptake of leucovorin and levels of N5,N10-methylenetetrahydrofolate achieved after leucovorin administration were similar in RP-MI-1788 and HS-42 cells, polyglutamylate forms of this coenzyme were less in the HS-42 cells as compared to RPMI-1788 cells. Based on these studies, the combination of trimetrexate with leucovorin should be further investigated as a way to increase the therapeutic index in some patients with soft tissue sarcomas.

Folate deficiency, biopterin and monoamine metabolism in depression.

Seven (21%) of 34 patients with a severe DSM-III diagnosis of major depression had red-cell folate levels below 150 ng/ml. This subgroup with folate deficiency had significantly lower CSF 5-hydroxyindoleacetic acid (5HIAA) compared to neurological controls. For all depressed patients red-cell folate was significantly correlated with CSF 5HIAA and homovanillic acid (HVA). CSF tetrahydrobiopterin (BH4) was significantly correlated with CSF 5HIAA and HVA and red-cell folate. Our observations provide further evidence of the links between folate, biopterin and monoamine metabolism in depression.

Evidence for pteridine regulation of lead-mediated inhibition of uroporphyrinogen and heme formation in rat bone marrow.

Uroporphyrin I (URO I) accumulation has been reported in the bone marrow of rats exposed to lead, suggesting a sensitivity of uroporphyrinogen III cosynthase (COSYN) to this heavy metal. Furthermore, it has been reported that a polyglutamated folate derivative may serve as a coenzyme for the catalytic action of hepatic uroporphyrinogen III cosynthase. These findings raised the question of whether depletion of polyglutamated folate could enhance the susceptibility of bone marrow COSYN to lead and potentially interfere with the formation of heme. Nitrous oxide, an anesthetic agent capable of causing bone marrow tetrahydrofolate deficiency, depressed total bone marrow polyglutamated folate content by 42% with significant reductions in all three chain lengths (5-7) identified in the bone marrow during an exposure period of 7 days at 4 hr/day. Lead acetate (15 mg/kg) administered by ip injection at Days 0 and 2 during a 7-day exposure to nitrous oxide resulted in an 84% increase of bone marrow URO I content, which was markedly higher than the increases of 22 and 38% seen with sole administration of lead or nitrous oxide, respectively. The combination of agents also produced a 48% rise in COPRO I, a 39 and 43% decrease in COPRO III and protoporphyrin, respectively, and a 42% decline in the activity of microsomal 7-ethoxycoumarin O-deethylase, which is hemoprotein, cytochrome P-450 mediated. Heme oxygenase activity was not altered by nitrous oxide, lead, or their combination. These results suggest that bone marrow folate deficiency may render COSYN more sensitive to lead as characterized by increased uroporphyrin I and coproporphyrin I isomer content, decreased coproporphyrin III and protoporphyrin content, and depressed microsomal hemoprotein, cytochrome P-450-mediated drug-metabolizing capability.