Determination of reduced folates in tumor and adjacent mucosa of colorectal cancer patients using LC‐MS/MS

A liquid chromatography electrospray ionization tandem mass spectrometry (LC‐MS/MS) method has been developed for the determination of 5,10‐methylenetetrahydrofolate (methyleneTHF), tetrahydrofolate (THF) and 5‐methyltetrahydrofolate (methylTHF) in colorectal mucosa and tumor tissues. The folate extraction method includes homogenization, heat and folate conjugase treatment to hydrolyze polyglutamyl folate to monoglutamyl folate. Before analysis on LC‐MS/MS, simple and fast sample purification with ultrafiltration (molecular weight cut‐off membrane, 10 kDa) was performed. Folates were detected and quantified using positive electrospray. The method described in the present paper was successfully applied to determine the level of three folate monoglutamates in mucosa and tumor samples from 77 colorectal cancer patients, starting from a limited amount of tissue. The results showed that the LC‐MS/MS method has a great advantage over other previously used methods because of its high sensitivity and selectivity. Significantly higher levels of methyleneTHF and THF were found in tumor compared with matched mucosa tissues. Folate levels in adjacent mucosa were associated with tumor location, age and gender. The correlation between folate levels and tumor site further strengthens the fact that development of right‐ and left‐sided tumors follows different pathways. Copyright © 2012 John Wiley & Sons, Ltd.     

An Improved Method to Assay Folates in Milk by a Turbidimetric Microbiological Assay

Abstract

Folates in milk are heat labile and methods used to protect these folates during sample preparation for microbiological estimation of this vitamin result in opaque solutions unsuitable for turbidimetric assays. This has necessitated the use of titrimetric assay for milk folates which are long and cumbersome. By the use of rennin precipitation of casein under conditions which preserve the folate activity, an optically clear solution is obtained which can then be used for turbidimetric assay. This method is described in this paper.     

Identification and determination of naturally occurring folates in grains of rice (Oryza sativa L.) by UPLC-MS/MS analysis

The genetic potential and biofortification of India-grown rice with bioavailable folate has not been studied yet. The objectives of this study were to determine the folates concentration in four cultivars of rice through UPLC–MS/MS. Total folate concentration in rice cultivars ranged from 11.0 to 51 μg/100 g with a mean of 26.0 μg/100 g. Among the four rice cultivars, the pigmented grain cultivar Nootripathu possesses two-fold rich sources of total folates than the other three non-pigmented grain cultivars. The average value of 100 g serving of rice grains could provide the amount of recommended daily allowance (% RDA) of dietary folates (6.5%) for adults, which ranged from 2.7–12.7%. Among the 5 individual forms of folates, 5-methyltetrahydrofolate was most abundant in rice cultivars followed by 10-Formylfolic acid and folic acid. The result of this study has been useful for biofortification of folates in rice.     

Determination of native folates in milk and other dairy products by high-performance liquid chromatography

Folates were measured in dairy products by high-performance liquid chromatography without prior sample clean-up. Detection limits for individual folates range from 0.3 to 7.3 ng/g. The folates were extracted from the sample matrix by adjusting the pH to 4.5 with acetic acid, centrifuging to remove precipitated proteins, and treating with conjugase to remove multiple polyglutamate residues. Folates were separated from other sample components using a reversed-phase column with a methanol-phosphate buffer (pH 6.8), and ion-pairing with tetrabutylammonium ion. Fluorescence was found to be the most useful detection technique. Fluorescence detection of reduced forms of the vitamin was achieved by post-column pH adjustment of the eluent with phosphoric acid, while the parent folic acid molecule required chemical oxidation with hypochlorite in order to obtain a fluorescent response.     

Application of liquid chromatography-electrospray ionisation mass spectrometry for determination of dietary folates: effects of buffer nature and mobile phase composition on sensitivity and selectivity

A sensitive and reliable liquid chromatography-mass spectrometry (LC-MS) method to determine dietary folates was developed and validated. Folates were detected and quantified using positive electrospray ionisation (ESI) with selective ion monitoring of protonated ions [M+H]+. The effects of buffer nature and mobile phase composition on separation, peak shape and intensity of MS signal were investigated. The acidic-basic properties of folates were successfully used to predict possible ionisation patterns, but they were not sufficient to predict the intensity of MS signal and the proportion of different ionisation products, which indicated that other parameters, such as gas phase acidity/basicity of analytes and ion evaporation mechanisms might be important. The use of aqueous acetic acid as volatile buffer was found to be preferable compared to formic acid due to considerable gain in intensity of MS signal for all folate forms studied. Limits of quantifications were 0.3 ng/mL for 5-methyltetrahydrofolate and 0.6 ng/mL for tetrahydrofolate, 10-formylfolic acid, 5-formyltetrahydrofolate and folic acid when using 20 microL injection. For 10-formylfolic acid, 5-formyltetrahydrofolate and folic acid the MS detection was found to be superior over commonly used fluorescence and UV detection in terms of selectivity and sensitivity. The method was successfully applied to analysis of folates in baker's yeast.     

A validated liquid chromatographic method for determining folates in vegetables, milk powder, liver, and flour

A liquid chromatographic (LC) method was elaborated for determining folates in foods. Folates were extracted by homogenizing in buffer and heat treatment. A portion was incubated with an enzyme preparation containing conjugase, amylase, and protease. After purification by affinity chromatography, folate monoglutamates were determined by reversed-phase LC with fluorescence and diode array detection. Gradient elution with phosphate buffer and acetonitrile was used to separate vitamers. The most abundant folate forms naturally present in foods were detected, including tetrahydrofolic acid, 5-methyltetrahydrofolic acid, and 5-formyltetrahydrofolic acid. 10-Formylfolic acid could be detected by applying a second fluorescence detector. Folic acid, used for fortification, might also be quantitated with this system. The difference between folate concentrations in sample extracts, with and without treatment of conjugase, is a measure of the quantity of polyglutamates in the food matrixes. An additional treatment with conjugase, amylase, and protease reflects the amount of matrix-bound folates. The LC system gave a linear response over the range 0-100 ng/mL. Detection limit for these compounds were 7 pg/mL for tetrahydrofolic acid and 5-methyltetrahydrofolic acid and 59 pg/mL for 10-formylfolic acid (signal-to-noise ratio > or = 3) when 100 microL was injected. Detection limits for 5-formyltetrahydrofolic acid and folic acid were 1 ng/mL. Repeatability relative standard deviation values for separate folates in 3 candidate Certified Reference Materials (CRMs)--mixed vegetables (CRM 485), pig liver (CRM 487), and whole-meal flour (CRM 121)--and a Certified Reference Material milk powder (CRM 421) varied from 3.3 to 21.0% for the concentration range 1.8-1440 micrograms/100 g. Recoveries ranged from 73 to 109%. Use of amylase and protease was advantageous. Use of a commercially available folate-binding protein for cleanup saved time and money and was effective. Results for 5-methyltetrahydrofolic acid were in good agreement with results obtained with other LC methods. Results for total folates were lower than results obtained with microbiological methods.     

Isotope ratio-based profiling of microbial folates

Folate metabolism, which is responsible for one-carbon transfer reactions in critical cellular processes including thymidine biosynthesis, is among the most important targets of antibiotic and anticancer drugs. Analysis of intracellular folates is complicated by three different types of folate modification: oxidation/reduction, methylation, and polyglutamylation. Here we present a method for quantifying the full diversity of intracellular folates by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The method begins with folate extraction using -75 degrees C methanol:water, with ascorbic acid and ammonium acetate added to prevent folate interconversion. The extract is then separated using hydrophilic interaction chromatography with an amino column, ionized by positive mode electrospray, and analyzed on a triple quadrupole instrument using multiple reaction monitoring. The method has been used to profile the folate pools in Escherichia coli and Saccharomyces cerevisiae, with absolute levels of selected folates in E. coli measured by spiking extracts of cells fed uniformly (13)C-glucose with purified, unlabeled folate standards. An isotope-ratio-based approach has been applied to study the effects of trimethoprim, a clinically important antibiotic that blocks bacterial dihydrofolate reductase. In addition to causing the expected increase in oxidized and decrease in reduced folates, trimethoprim triggered a dramatic and previously unrecognized shift towards shorter polyglutamate chain lengths. This finding highlights the potential for analysis of the full spectrum of cellular folates by MS/MS to unveil novel biological phenomena.     

Direct determination of folate monoglutamates in plasma by high-performance liquid chromatography using an automatic precolumn-switching system as sample clean-up procedure

A simple and rapid technique for the simultaneous isolation and analysis of folate monoglutamates (folic acid, 7,8-dihydrofolic acid, 5,6,7,8-tetrahydrofolic acid and 5-formyl-, 5-methyl- and 10-formyl-5,6,7,8-tetrahydrofolic acids) was developed using reversed-phase high-performance liquid chromatography with an automatic precolumn-switching system. The plasma or the dissolved diet samples were directly injected onto a short precolumn flushed with 50 mM phosphate buffer. The folate vitamers absorbed on the precolumn were backflushed onto the analytical column with a 25 mM phosphate buffer containing 5% methanol and then detected by UV absorption at 280 nm. A linear response was found between the injected sample amounts and the integrated areas for all vitamers analysed. The detection limit was 1-10 pmol and the precision ranged from 1.6 to 10%, depending on the metabolite studied. The recovery rates of folates in plasma were 90-95%. Decomposition of the unstable folates was avoided. Our method was applied to the analysis of mouse plasma and animal diets.     

Determination of folates in foods by high-performance liquid chromatography with fluorescence detection after precolumn conversion to 5-methyltetrahydrofolates

A liquid chromatographic-fluorimetric determination of folates in foodstuffs including their extraction, without or with deconjugation, chemical conversion to 5-CH3-H4PteGlu(n) and purification of the extract by affinity chromatography is reported. The conversion enables the analysis of total folates and also of the contents of the different mono- and polyglutamate forms of the folates. The method has a satisfactory day-to-day repeatability (never,more than 10%) and a very low detection limit (0.02 pmol per injection). Depending on the folate studied, the recovery rates varied from 78% (10-CHO-PteGlu) to 98% (5-CHO-H4PteGlu). Furthermore it has been possible to show that the deconjugation of the folates by rat plasma conjugase was incomplete in foodstuffs whereas chicken pancreas conjugase effectively converted the different folate polyglutamates into folate diglutamates. It could not be demonstrated that prior hydrolysis with a protease and amylase was useful for the analysis of the different foodstuffs studied (yeast, spinach, beef liver, beef fillet and peas) when deconjugation was performed with the chicken pancreas conjugase.     

LC/MS/MS identification of some folic acid degradation products after E-beam irradiation

Folates belong to the B vitamin group based on the parental compound folic acid (FA). They are involved in important biochemical processes like DNA synthesis and repair. FA is composed of a pteridine ring, p-aminobenzoic acid and glutamate moieties. The human metabolism is not able to synthesize folates and therefore obtain them from diet. FA, a synthetic vitamin, is used as a food fortificant because of its low price, relative stability and increased bioavailability compared to natural folate forms. FA is known to be a sensitive compound easily degradable in aqueous solution by ultraviolet and visible light towards various by-products. Irradiation is a process for preservation of foods that uses accelerated electrons, gamma rays or X-rays. Irradiation is proposed for the treatment of various food products, eliminating or reducing pathogens and insects, increasing the storage time and replacing chemical fumigants. This study concerns the identification of degradation products of FA after E-beam irradiation. FA aqueous solutions were irradiated with a Van de Graaff electrons beam accelerator (2 MeV, 100 μA current, 20 cm scan width, dose rate about 2 kGy/s). Applied doses were between 0 (control) and 10.0 kGy. Absorbed doses were monitored with FWT 60.00 radiochromic dosimeters.     

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%).     

Folate Content and Yolk Color of Hen Eggs from Different Farming Systems

This study aimed to compare folate contents in hen eggs from four different farming systems, namely organic, free range, barn, and cage one. Folate retention during egg boiling was studied as well. The contents of individual folate vitamers were determined using the high-performance liquid chromatography method (HPLC), following trienzyme treatment. Folate content in eggs differed significantly (p < 0.05) due to the rearing system, with the highest mean content determined in the eggs from organic farming (113.8 µg/100 g). According to this study, one egg (60 g) may provide 40–86 µg of folates, which corresponds to 10–22% of the recommended daily intake for adults, 400 µg according to the Nutrition Standards for the Polish Population. The predominant folate form found in egg was 5-methyltetrahydrofolate, which showed considerably greater stability under boiling compared to 10-formylfolic acid present in a lower amount. In most eggs tested, the losses in total folate content did not exceed 15%. The color of yolk of the most folate-abundant organic eggs, had the highest value of lightness (L*) and the lowest value of redness (a*). This, however, does not correspond to consumer preferences of intense golden yolk color.     

Rapid radio-proteinbinding measurement of urine folates

A method is presented for the determination of urine folates by a commercial radio-proteinbinding assay widely used in clinical diagnostics for assessing plasma folate levels. Good recovery of urine folates over the range of the calibration curve was observed. Because of the speed, sensitivity and precision of this test, urine folate levels can be measured in bioavailability studies or for the assessment of folate status.     

Assay of whole blood (6S)-5-CH3-H4folate using ultra performance liquid chromatography tandem mass spectrometry

Folates act as essential coenzymes in many biological pathways, including the synthesis and methylation of DNA. Low folate concentration in serum and whole blood (WB) is associated with several disease conditions. We describe a stable-isotope-dilution ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for the quantification of (6S)-5-CH(3)-H(4)folate (where H(4)folate is tetrahydrofolate) and non-CH(3)-H(4)folate [sum of HCO-H(4)folate, (6R)-5,10-CH(+)-H(4)folate, (6R)-5,10-CH(2)-H(4)folate, (6S)-H(4)folate, dihydrofolate, and folic acid] in WB. The assay includes a solid-phase extraction procedure after the hemolysis and deconjugation. The method was linear over the concentration range from 0.2 to 200 nmol/L. The limits of detection were 0.40 nmol/L or lower for the folate forms. The interassay coefficients of variation were 7.4% for (6S)-5-CH(3)-H(4)folate and 15.4% for non-CH(3)-H(4)folate. For the folate forms, the recoveries were between 97.1% and 102.7%. Sample preparation caused the generation of artificial folic acid in WB and serum in a dose-dependent manner, which can lead to misinterpretation of the results. The use of antioxidants could not prevent the formation of folic acid. The median fasting WB folate concentrations from 42 nonsupplemented and nonfortified adults were 576 nmol/L (6S)-5-CH(3)-H(4)folate and 73.6 nmol/L non-CH(3)-H(4)folate, and 1,206 nmol/L (6S)-5-CH(3)-H(4)folate and 155 nmol/L non-CH(3)-H(4)folate for 35 adults who had taken 500 μg of folic acid, 50 mg of vitamin B(6), and 500 μg of vitamin B(12) per day orally for 6 months. In conclusion, the UPLC-MS/MS method is fast and has a good sensitivity and selectivity for WB folates. We observed a dose-dependent oxidation of (6S)-H(4)folate, which resulted in the formation of artificial folic acid in serum and WB. To minimize this effect, we recommend a fast sample preparation.     

The Self-Recognition and Self-Assembly of Folic Acid Salts in Isotropic Water Solution

The self-assembly of alkaline folates in isotropic water solutions, with or without added salts, has been investigated by small-angle neutron-scattering, circular-dichroism, and NMR techniques. The assembled species are chiral, cylindrical aggregates of finite length, composed of stacked tetramers; each tetramer is formed by Hoogsteen-bonded folate residues. The assembly process is more efficient in the presence of an excess of Na^I ions, leading to longer aggregates with stronger tetramer-tetramer-tetramer interaction. In pure water, the rods are shorter and the tetramer-tetramer interaction weaker. Association between folates can be detected by circular-dichroism spectroscopy starting from a concentration of 6· 10^?4 mol l^?1, well below the critical concentration for the formation of the cholesteric mesophase (ca. 0.5 mol l^?1).     

2，3－二甲基－1－对氯苯磺酰基咪唑啉盐与邻氨基苯酚反应的量子化学研究

用量子化学方法对叶酸辅酶模型化合物2，3－二甲基－1－对氯苯磺酰基咪唑 啉盐与邻氨基苯酚的反应进行了理论研究。结果表明，咪唑啉环有两种开环方式， 反应可以通过两种途径实现，得到较稳定的中间体或者实现一碳单元的完全转移。 通过优化计算所有步骤的中间体和过渡态的结构可知，各个中间体和过渡态具有不 同的构型、构象，有些过程的构象变化是进行下一步反应所必需的，在所有过程中 质子转移步骤过渡态的能量最高，是反应的限速步骤。     

Novel functional hyperbranched polyether polyols as prospective drug delivery systems

Multifunctional hyperbranched polyether polyols bearing protective poly(ethylene glycol) (PEG) chains with or without the folate targeting ligand at their end have been prepared. Solubilization in these polymers of a fluorescent probe, pyrene, and an anticancer drug, tamoxifen, was physicochemically investigated. It was found that PEG chains attached at the surface of these hyperbranched polymers, in addition to their well-established protective role, enhance the encapsulation efficiency of the polymers. The release of pyrene and tamoxifen observed upon addition of sodium chloride is, in most of the cases, significant only at concentrations exceeding the physiological extracellular concentration. Thus, a significant amount of the probe or drug remains solubilized inside the carriers, which is an encouraging result if the polymers are to be used for drug delivery.     

Comprehensive metabolic profiling of mono- and polyglutamated folates and their precursors in plant and animal tissue using liquid chromatography/negative ion electrospray ionisation tandem mass spectrometry

This work reports the use of reversed-phase ion-pair chromatography coupled to electrospray ionisation mass spectrometry for the simultaneous profiling of folate-based metabolites including natural folates, their polyglutamatyl derivatives and their biosynthetic precursors in plant and animal tissue. A simple sample preparation method, using 0.1% citric acid and ascorbic acid in ice-cold methanol, was used to extract and stabilise the folates, and three internal standards were used. Chromatography was on a C18 column using slow gradient elution with a mobile phase consisting of methanol/water with 5 mM dimethylhexylamine. Mass spectrometric detection was performed by multiple reaction monitoring in seven separate time windows in negative ion mode over the 25 min run time. Full, quantitative analysis was obtained for 16 folates and a 'semi-quantitative' analysis was possible for all other folates with up to eight conjugated glutamate residues by reference to structurally related calibration standards. The precision, accuracy and recovery of the method were generally within the accepted guidelines for a quantitative bioanalytical method and the method was linear over the range 0.2 to 10 ng of individual folate per sample. The method was applied to profile mono- and polyglutamated tetrahydrofolates (including subcellular analysis) in a range of plant species, including Arabidopsis, spinach, Brassica and wheat; the technique was also successfully applied to the profiling of folates in mouse tissue.     

Further Studies on the Interaction of Immobilized Folate Binding Protein and Enzyme-Folate Conjugates; An Enzyme-Linked Competitive Binding Assay for Folate

Abstract

The kinetic and thermodynamic properties of the folate interaction with immobilized folate binding protein (FBP) are examined. The use of enzyme-rather than radio-labeled folate provides insight into the complex binding mechanism of folate with FBP and indicates that polymerization of the binding protein (evident for FBP-folate association in solution) is not a prerequisite for the cooperative behavior observed. An enzyme-linked competitive binding assay for folate based on this interaction is described and dose-response curves demonstrate the sensitivity and selectivity of the method. The accuracy of the assay is tested by determining folate in infant formula.     

Microbiological assay-trienzyme procedure for total folates in cereals and cereal foods: collaborative study

In 1996, U.S. Food and Drug Administration regulations mandated the fortification of enriched cereal-grain products with folic acid, thereby emphasizing the need for validated methods for total folates in foods, particularly cereal products. The AOAC Official Methods (944.12, 960.46) currently used for the analysis of folate in foods for compliance purposes are microbiological methods. When the fortification regulations were finalized, no Official AOAC or Approved AACC methods for folate in cereal-grain products were in place. The AOAC Official Method (992.05) for folic acid in infant formula does not incorporate important improvements in the extraction procedure and was not considered suitable for the analysis of folates in foods in general. A microbiological assay protocol using a trienzyme extraction procedure was prepared and submitted for comments to 40 laboratories with recognized experience in folate analysis. On the basis of comments, the method was revised to have the conjugase (gamma-glutamyl-carboxy-peptidase) treatment follow a protease treatment, to include the use of cryoprotected inoculum, and to include the spectroscopic standardization of the standard and optional use of microtiter plates. Thirteen laboratories participated in a collaborative study of 10 required and 10 optional cereal-grain products, including flour, bread, cookies, baking mixes, and ready-to-eat breakfast cereals. The majority of the participating laboratories performed the assay by the standard test tube method; others used the microtiter plate modification for endpoint quantitation with equal success. For the required products, the relative standard deviation between laboratories (RSD(R)) ranged from 7.4 to 21.6% for 8 fortified (or enriched) products compared with expected (Horwitz equation-based) values of 11-20%. RSD(R) values were higher (22.7-52.9%) for 2 unfortified cereal-grain products. For the optional products, the RSD(R) ranged from 1.8 to 11.2% for 8 fortified products. RSD(R) values were higher (27.9-28.7%) for 2 unfortified cereal-grain products. Based on the results of the collaborative study, the microbiological assay with trienzyme extraction is recommended for adoption as Official First Action.