Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) for the sensitive determination of folates in rice.

High-performance liquid chromatography, coupled to tandem mass spectrometry (HPLC-MS/MS) has been established as the method of choice for the sensitive and simultaneous determination of different folates in a particular matrix, especially when only minute quantities of material are available. Using a previously developed and validated HPLC-MS/MS method as a starting point, we here report on the development and validation of an ultra-performance liquid chromatography (UPLC-MS/MS) method for analysis of folates in rice, which allows higher throughput and better resolution. UPLC was performed under gradient conditions on an Acquity HSS T3 column, followed by tandem mass spectrometry detection.

C1 metabolism and chlorophyll synthesis: the Mg-protoporphyrin IX methyltransferase activity is dependent on the folate status.

* Tetrahydrofolate derivatives are central cofactors of C1 metabolism. Using methotrexate as a specific inhibitor of folate biosynthesis, we altered the folate status in 10-d-old etiolated pea (Pisum sativum) leaves and followed the rate of chlorophyll synthesis upon illumination. * In our conditions, the folate concentration decreased only from 5.

Optimisation and validation of a liquid chromatography-tandem mass spectrometry method for folates in rice.

An LC-MS/MS method has been developed and validated for the analysis of six monoglutamate folates in rice. Accurate determination of folates in rice seeds required the optimisation of an extraction procedure encompassing homogenisation, heat treatment, tri-enzyme treatment (alpha-amylase, protease and deconjugase), centrifugation and ultrafiltration. The resulting extract was loaded onto a Polaris C 18-A column, followed by gradient elution and detection by tandem mass spectrometry.

A genome-wide and metabolic analysis determined the adaptive response of Arabidopsis cells to folate depletion induced by methotrexate.

Control of folate homeostasis is essential to sustain the demand for one-carbon (C1) units that are necessary for major biological functions, including nucleotide synthesis and methylation reactions. In this study, we analyzed the genome-wide and metabolic adaptive response of Arabidopsis (Arabidopsis thaliana) cells to folate depletion induced by the antifolate methotrexate. Drug treatment induced a response typical to xenobiotic stress and important changes in folate content and composition.

Folate fortification of rice by metabolic engineering.

Rice, the world's major staple crop, is a poor source of essential micronutrients, including folates (vitamin B9). We report folate biofortification of rice seeds achieved by overexpressing two Arabidopsis thaliana genes of the pterin and para-aminobenzoate branches of the folate biosynthetic pathway from a single locus. We obtained a maximal enhancement as high as 100 times above wild type, with 100 g of polished raw grains containing up to four times the adult daily folate requirement.

pH stability of individual folates during critical sample preparation steps in prevision of the analysis of plant folates.

The stability and eventual interconversion of nine mono-glutamate folates (5-methyl-tetrahydrofolate, tetrahydrofolate, 5-formyltetrahydrofolate, 5,10-methenyltetrahydrofolate, 5,10-methylenetetrahydrofolate, dihydrofolate, 10-formylfolic acid, 10-formyltetrahydrofolate and folic acid) during the typical sample preparation steps (heat treatment for 10 min at 100 degrees C and incubation for 2 h at 37 degrees C) at different pH values have been investigated by LC-MS/MS. An LC-MS/MS method with isotopically labelled [(13)C(5)]5-methyltetrahydrofolate and [(13)C(5)] folic acid as internal standards has been developed with enhanced sensitivity using a Chromolith RP-18 column. 5-Methyltetrahydrofolate, folic acid and 10-formylfolic acid are relatively stable at different pHs (from 2 to 10) with and without heat treatment.

Cytosolic hydroxymethyldihydropterin pyrophosphokinase/dihydropteroate synthase from Arabidopsis thaliana: a specific role in early development and stress response.

In plants, 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase/7,8-dihydropteroate synthase (mitHPPK/DHPS) is a bifunctional mitochondrial enzyme, which catalyzes the first two consecutive steps of tetrahydrofolate biosynthesis. Mining the Arabidopsis genome data base has revealed a second gene encoding a protein that lacks a potential transit peptide, suggesting a cytosolic localization of the isoenzyme (cytHPPK/DHPS). When the N-terminal part of the cytHPPK/DHPS was fused to green fluorescent protein, the fusion protein appeared only in the cytosol, confirming the above prediction.