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Michael Rychlik - One of the best experts on this subject based on the ideXlab platform.

  • assessing volumetric absorptive microsampling coupled with stable isotope dilution assay and liquid chromatography tandem mass spectrometry as potential diagnostic tool for whole blood 5 Methyltetrahydrofolic Acid
    Frontiers in Nutrition, 2017
    Co-Authors: Markus Kopp, Michael Rychlik
    Abstract:

    Volumetric absorptive microsamplers (VAMS) have been developed recently as a promising tool for clinical blood sampling. Compared to dried blood spot (DBS) samples analysed by accurate stable isotope dilution assays (SIDAs) the new technique could provide further substantial miniaturizing of folate assays by eliminating hematocrit effects and uneven analyte distribution within the sample. Herein we present a miniaturized SIDA coupled with LC-MS/MS measurement of 5-Methyltetrahydrofolic Acid as main folate vitamer in whole blood using [13C5]-5-Methyltetrahydrofolic Acid as internal standard. Elution and extraction of only 10.8 µL dried whole blood was carried out by centrifugation followed by enzymatic treatment for polyglutamate deconjugation. Matrix separation was achieved by heating and centrifugation. To proof applicability we screened 11 volunteers for their whole blood folate status. Limits of detection and quantitation were 9 and 26 nmol∙L-1 and, therefore, sufficiently low for screening folate status. Recoveries were 97 (±5.8), 99 (±2.8) and 96 (±6.1)% for 800, 400 and 200 nmol∙L-1 5-Methyltetrahydrofolic Acid, respectively. Precision of the LC-MS/MS instrument and inter-assay precision trials revealed CVs of 8.1 and 3.5% (294 nmol∙L-1), respectively, thus confirming reproducible and precise quantitation. Compared to fresh whole blood no significant degradation of 5-methyltetrahydrofolate was observed after 2.5 h drying at room temperature. VAMS 5-CH3-H4folate was stable for at least 3 weeks at -20°C. In our pilot study accurate and diagnostically conclusive determination of folate status was verified. Nevertheless, blood sampling should be performed by trained individuals to avoid substantial errors concerning the absorbed volume. Endogenous folate in rat serum and chicken pancreas caused a significant background especially at low blood 5-CH3-H4folate levels and, thus, removing these background folates or alternative mixtures for polyglutamate deconjugation are needed. The superior feasibility of a minimized blood collection with VAMS allows further progress regarding time- and cost-effective methodologies in newborn or population screenings for 5-methyltetrahydrofolate status. Further steps towards minimization could include an automated assay coupled with UPLC-MS/MS.

  • Measurements of Intra- and Extra-Cellular 5-Methyltetrahydrofolate Indicate that Bifidobacterium Adolescentis DSM 20083T and Bifidobacterium Pseudocatenulatum DSM 20438T Do Not Actively Excrete 5-Methyltetrahydrofolate In vitro
    Frontiers in Microbiology, 2017
    Co-Authors: Markus Kopp, Kerstin Dürr, Matthias Steigleder, Thomas Clavel, Michael Rychlik
    Abstract:

    Certain intestinal bifidobacteria have the ability to synthesize folates. In vitro experiments revealed a high production, cellular accumulation, and release of reduced folate vitamers like 5-methyltetrahydrofolate and tetrahydrofolate in folate-free medium (FFM). However, it is still unclear to which extent synthesized folates are polyglutamylated and probably not available for transport, and if they are actively released by excretion. To address these questions, we characterized intra- and extracellular pteroylmonoglutamates and polyglutamylated 5-methyltetrahydrofolate (5-CH3-H4PteGlu2-4) in B. adolescentis DSM 20083T and B. pseudocatenulatum DSM 20438T in vitro. Folates were measured by means of stable isotope dilution assays coupled with LC-MS/MS analysis using [2H4]-5-Methyltetrahydrofolic Acid, [2H4]-tetrahydrofolic Acid and [2H4]-5-formyltetrahydrofolic Acid as internal standards. Cell viability was examined by fluorescence microscopy. Quantitation of folate production by B. adolescentis during the stationary phase revealed a linear increase of dead cells paralleled by increasing concentration of 5-formyltetrahydrofolate and 5-methyltetrahydrofolate (100% 5-CH3-H4PteGlu4) in FFM, whereas the intracellular concentrations of these vitamers remained constant. After 24 h, B. adolescentis (125 mg cells, wet weight) produced a total amount of 0.846 nmol 5-CH3-H4folate: 0.385±0.059 nmol (46±7%) and 0.461±0.095 nmol (54±11%) measured in the intracellular (viable cells; 52±3% measured by fluorescence microscopy) and extracellular (lysed cells; 48±3%) fraction, respectively. For B. pseudocatenulatum (124 mg cells, wet weight), 1.135 nmol 5-CH3-H4folate was produced after 24 h, and a similar proportionality between intra/extracellular folate concentrations and viable/lysed cells was observed. These results indicate that the strains tested produce and accumulate 5-CH3-H4PteGlu4 for cellular metabolism, and that extracellular concentrations of the vitamer arise from cell lysis.

  • development of stable isotope dilution assays for the quantitation of intra and extracellular folate patterns of bifidobacterium adolescentis
    Journal of Chromatography A, 2016
    Co-Authors: Markus Kopp, Kerstin Dürr, Matthias Steigleder, Thomas Clavel, Michael Rychlik
    Abstract:

    Folate-producing bifidobacteria have been studied extensively but appropriate methods for detailed quantitation of intra- and extracellular pteroylmono- and pteroylpolyglutamate patterns are lacking. Therefore, B. adolescentis DSM 20083T was cultivated in folate-free medium (FFM) for 24h to develop and validate stable isotope dilution assays (SIDAs) coupled with LC-MS/MS for the determination of 5-formyltetrahydrofolic Acid (5-HCO-H4folate), 10-formylfolic Acid (10-HCO-PteGlu), tetrahydrofolic Acid (H4folate), folic Acid (PteGlu) and 5-Methyltetrahydrofolic Acid (5-CH3-H4folate) including its di-, tri-, and tetraglutamic vitamers (5-CH3-H4PteGlu2-4). The respective monoglutamylated isotopologues labelled with deuterium were used as internal standards for quantitation. Limits of detection and quantitation (LOD/LOQ) were sufficiently low to quantify 48.2nmol L-1 5-CH3-H4folate (5.7/17nmolL-1) and 71.0nmolL-1 5-HCO-H4folate (10/30nmolL-1) as major folate vitamers extracellularly and 124nmolL-1 5-CH3-H4folate (3.4/10nmolL-1), 213nmolL-1 5-HCO-H4folate (4.8/14nmolL-1), and 61.4nmolL-1 H4folate (2.3/7.0nmolL-1) intracellularly after deconjugation. The major portion of native 5-CH3-H4folate vitamer was ascribed to its tetraglutamate ( > 95%). Concentrations of mono-, di-, tri-, and pentaglutamylated folates were below LOD or LOQ. Intra-assay precision coefficients of variation (CVs) ranged from 7% (at a concentration of 53.9nmolL-1 for 5-CH3-H4PteGlu4), 15% (25.5nmolL-1 5-CH3-H4folate) to 18% (78.5nmolL-1 5-HCO-H4folate), extracellularly, and from 6% (60.7nmolL-1 5-CH3-H4PteGlu4), 7% (202nmolL-1 5-HCO-H4folate), 10% (67.1nmolL-1 H4folate) to 11% (127nmolL-1 5-CH3-H4folate), intracellularly. Inter-assay precision CVs ranged from 2% (54.7nmolL-1 5-CH3-H4PteGlu4), 3% (71nmolL-1 5-HCO-H4folate) to 11% (48.2nmolL-1 5-CH3-H4folate), extracellularly, and from 1% (61.4nmolL-1 H4folate), 5% (213nmolL-1 5-HCO-H4folate), 6% (63.5nmolL-1 5-CH3-H4PteGlu4) to 10% (124nmolL-1 5-CH3-H4folate), intracellularly, thus showing excellent reproducibility. Recoveries for all analytes under study ranged between 81 and 113%. These newly developed methods enable reproducible, precise and sensitive quantitation of eight bacterially synthesized folate vitamers in two totally different matrices, including both monoglutamates and polyglutamates. Furthermore, we here present the first assay using solely monoglutamylated [2H4]-5-CH3-H4folate to quantify native polyglutamate patterns of this vitamer in bacteria which might replace time-consuming determination of monoglutamates in the future.

  • RESEARCH ARTICLE Quantitation of 5-Methyltetrahydrofolic Acid in Dried Blood Spots and Dried Plasma Spots by Stable Isotope Dilution Assays
    2016
    Co-Authors: Michael Rychlik
    Abstract:

    Because of minimal data available on folate analysis in dried matrix spots (DMSs), we com-bined the advantages of stable isotope dilution assays followed by LC-MS/MS analysis with DMS sampling to develop a reliable method for the quantitation of plasma 5-methyltetrahy-drofolic Acid in dried blood spots (DBSs) and dried plasma spots (DPSs) as well as for the quantitation of whole blood 5-Methyltetrahydrofolic Acid in DBSs. We focused on two diag-nostically conclusive parameters exhibited by the plasma and whole blood 5-methyltetrahy-drofolic Acid levels that reflect both temporary and long-term folate status. The method is performed using the [2H4]-labeled isotopologue of the vitamin as the internal standard, and three steps are required for the extraction procedure. Elution of the punched out matrix spots was performed using stabilization buffer including Triton X-100 in a standardized ultrasonication treatment followed by enzymatic digestion (whole blood only) and solid-phase extraction with SAX cartridges. This method is sensitive enough to quantify 27 nmol/ L whole blood 5-Methyltetrahydrofolic Acid in DBSs and 6.3 and 4.4 nmol/L plasma 5-Methyltetrahydrofolic Acid in DBSs and DPSs, respectively. The unprecedented accurate quantification of plasma 5-Methyltetrahydrofolic Acid in DBSs was achieved by thermal treatment prior to ultrasonication, inhibiting plasma conjugase activity. Mass screenings are more feasible and easier to facilitate for this method in terms of sample collection and stor-age compared with conventional clinical sampling for the assessment of folate status

  • Quantitation of 5-Methyltetrahydrofolic Acid in Dried Blood Spots and Dried Plasma Spots by Stable Isotope Dilution Assays.
    PloS one, 2015
    Co-Authors: Markus Kopp, Michael Rychlik
    Abstract:

    Because of minimal data available on folate analysis in dried matrix spots (DMSs), we combined the advantages of stable isotope dilution assays followed by LC-MS/MS analysis with DMS sampling to develop a reliable method for the quantitation of plasma 5-Methyltetrahydrofolic Acid in dried blood spots (DBSs) and dried plasma spots (DPSs) as well as for the quantitation of whole blood 5-Methyltetrahydrofolic Acid in DBSs. We focused on two diagnostically conclusive parameters exhibited by the plasma and whole blood 5-Methyltetrahydrofolic Acid levels that reflect both temporary and long-term folate status. The method is performed using the [2H4]-labeled isotopologue of the vitamin as the internal standard, and three steps are required for the extraction procedure. Elution of the punched out matrix spots was performed using stabilization buffer including Triton X-100 in a standardized ultrasonication treatment followed by enzymatic digestion (whole blood only) and solid-phase extraction with SAX cartridges. This method is sensitive enough to quantify 27 nmol/L whole blood 5-Methyltetrahydrofolic Acid in DBSs and 6.3 and 4.4 nmol/L plasma 5-Methyltetrahydrofolic Acid in DBSs and DPSs, respectively. The unprecedented accurate quantification of plasma 5-Methyltetrahydrofolic Acid in DBSs was achieved by thermal treatment prior to ultrasonication, inhibiting plasma conjugase activity. Mass screenings are more feasible and easier to facilitate for this method in terms of sample collection and storage compared with conventional clinical sampling for the assessment of folate status.

Christine M Pfeiffer - One of the best experts on this subject based on the ideXlab platform.

  • EW. Determination of folate vitamers in human serum by stable-isotope-dilution tandem mass spectrometry and comparison with radioassay and microbiologic assay. Clin Chem 2004;50:423–32
    2015
    Co-Authors: Christine M Pfeiffer, Zia Fazili, Les Mccoy, Ming Zhang, Elaine W Gunter
    Abstract:

    Background: Current clinical methods for folate give different results and cannot measure the various forms of folate. We developed an isotope-dilution tandem mass spectrometric method coupled to liquid chroma-tography (LC/MS/MS) as a candidate reference method for 5-Methyltetrahydrofolic Acid (5MeTHF), 5-formyltet-rahydrofolic Acid (5FoTHF), and folic Acid (FA) in hu-man serum. Methods: We quantitatively isolated folates from 275 L of serum with a phenyl solid-phase extraction car-tridge, then detected and quantified them in stabilized serum extracts by positive-ion electrospray ionization LC/MS/MS. We used an isocratic mobile phase of acetic Acid in organic solvent on a C8 analytical column. 13C-labeled folates were used as internal standards. Results: Limits of detection in serum were 0.13 (5MeTHF), 0.05 (5FoTHF), and 0.07 (FA) nmol/L. Within-and between-run imprecision (CV) was <7 % for 5MeTHF and <10 % for 5FoTHF at concentrations>0.5 nmol/L, and <10 % for FA at concentrations>2.0 nmol/L. Total folate (TFOL) concentrations determined by com-petitive protein binding RIA were 9 % lower than results obtained with LC/MS/MS. The microbiologic assay gave 15 % higher TFOL results with FA calibra-tor and no difference with 5MeTHF calibrator. The mean (SD) [range] TFOL in 42 sera was 35.5 (17.8) [6.5–75.6] nmol/L. Thirty-two samples with TFOL <50 nmol/L had, on average, 93.3 % 5MeTHF, 2.3 % FA, and 4.4 % 5FoTHF. Ten samples with TFOL>50 nmol/L had

  • influence of 5 10 methylenetetrahydrofolate reductase polymorphism on whole blood folate concentrations measured by lc ms ms microbiologic assay and bio rad radioassay
    Clinical Chemistry, 2008
    Co-Authors: Zia Fazili, Christine M Pfeiffer, Mindy Zhang, Ram B Jain, Deborah Koontz
    Abstract:

    Background: The 5,10-methylenetetrahydrofolate reductase (NADPH) ( MTHFR ) C677T polymorphism may affect whole-blood folate pattern measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and total folate measured by LC-MS/MS, microbiologic assay, and Bio-Rad radioassay (BR). Methods: We analyzed 171 whole blood hemolysates from 2 blood banks for folate pattern and total folate concentrations using these 3 methods and determined MTHFR genotype. Results: The median (range) total folate concentration by LC-MS/MS was higher in the US set [378 (228–820) nmol/L; n = 96] than in the European set [250 (122–582) nmol/L; n = 75]. The whole-blood folate pattern [median (range)] was similar for individuals with C/C (n = 73) and C/T (n = 66) genotype: 88% (71%–91%) and 86% (50%–91%), respectively, for 5-Methyltetrahydrofolic Acid (5CH3THF) vs 12% (9%–29%) and 14% (9%–51%) for forms other than 5-Methyltetrahydrofolic Acid (non-5CH3THF). Individuals with T/T (n = 32) genotype had 58% (22%–87%) 5CH3THF vs 42% (13%–78%) non-5CH3THF. Compared with microbiologic assay results, LC-MS/MS ( r = 0.94) and BR ( r = 0.87) results were significantly lower (−10% and −45%, respectively); however, these differences were concentration dependent and also genotype dependent for the BR assay (−48% for C/C+C/T and −31% for T/T). The microbiologic assay completely recovered [mean (SD)] folates added to a whole blood hemolysate, except for tetrahydrofolic Acid (THF) [46.4% (8.1%)]. The BR assay underrecovered 5CH3THF [51% (4.1%)] and 5-formyltetrahydrofolic Acid [18% (0.1%)], and overrecovered THF [152% (19%)]. Conclusion: MTHFR C677T polymorphism influences the folate pattern in whole blood. The agreement between total folate by LC-MS/MS and microbiologic assay, independent of the MTHFR genotype, allows the use of one regression equation. Because BR results are genotype dependent, different regression equations should be used.

  • comparison of serum folate species analyzed by lc ms ms with total folate measured by microbiologic assay and bio rad radioassay
    Clinical Chemistry, 2007
    Co-Authors: Zia Fazili, Christine M Pfeiffer, Mindy Zhang
    Abstract:

    Background: The Bio-Rad QuantaPhase II radioassay (BR), used for 25 years to measure total folate (TFOL) concentrations for the National Health and Nutrition Examination Survey (NHANES), will be discontinued in 2007. Liquid chromatography–tandem mass spectrometry (LC-MS/MS) or a microbiologic assay (MA) will be used in the future. Methods: We measured folate species by LC-MS/MS and TFOL by MA and BR in 327 serum samples. Results: LC-MS/MS measured 5-Methyltetrahydrofolic Acid (5CH3THF; 82%), folic Acid (FA; 8%), 5-formyltetrahydrofolic Acid (5CHOTHF; 6%), tetrahydrofolic Acid (THF; 4%), and 5,10-methenyltetrahydrofolic Acid (5,10CH=THF; 0%). The sum of the folate species correlated well with TFOL measured by MA ( R 2 = 0.97) and BR ( R 2 = 0.91). Compared with LC-MS/MS results, MA and BR values were significantly lower (−6% and −29%, respectively); however, these differences were concentration dependent. The MA almost completely recovered folates added to serum samples except for FA [69% (3%)] and THF [36% (10%)]. The BR underrecovered 5CH3THF [61% (9%)] and 5CHOTHF [38% (14%)] and overrecovered 5,10CH=THF [234% (32%)]. Multiple linear regression models with log-transformed data yielded a good fit for converting BR data to MA or LC-MS/MS data and MA data to LC-MS/MS data. Conclusions: The good correspondence between the sum of folate species determined by LC-MS/MS and TFOL determined by MA makes these 2 assays interchangeable. The BR produces much lower results, on average, probably because of 5CH3THF underrecovery. The conversion equations provided could be used for future NHANES time trend analyses.

  • measurement of folates in serum and conventionally prepared whole blood lysates application of an automated 96 well plate isotope dilution tandem mass spectrometry method
    Clinical Chemistry, 2004
    Co-Authors: Zia Fazili, Christine M Pfeiffer
    Abstract:

    Folate nutriture is associated with neural tube defects (1), vascular diseases (2), certain forms of cancer (3), and cognitive function (4). To reduce the risk for neural tube defects, the US Food and Drug Administration mandated folate fortification of cereal grain products beginning in January 1998 (5). Serum folate and whole blood folate (WBF) are measured to determine folate status. Clinical methods to determine serum folate and WBF, such as the microbiologic assay and various immunoassays, measure only total folate (TF) and present variable results, especially for WBF (6)(7). Few methods have described the measurement of WBF by chromatography-based methods (8)(9)(10)(11)(12)(13)(14)(15). Gas chromatography/mass spectrometry used to analyze WBF after cleavage of folates to p -aminobenzoic Acid showed greater sensitivity than previous chromatographic methods and used for the first time an isotope-labeled internal standard (IS). The method, however, requires a complex and lengthy multistep sample preparation including chemical derivatization (11)(13)(14)(15). We describe here the first automated 96-well plate stable-isotope-dilution liquid chromatography–tandem mass spectrometry (LC/MS/MS) method that measures intact folate monoglutamates in conventionally prepared whole blood (WB) lysates and serum. To increase sample throughput and minimize the extent of manual sample preparation, we adapted our recently developed manual solid-phase extraction (SPE) LC/MS/MS method for 5-Methyltetrahydrofolic Acid (5CH3THF), 5-formyltetrahydrofolic Acid (5CHOTHF), and folic Acid (FA) in serum (16) to an automated method using 96-well plates. In addition, we extended this method to measure additional folate forms in WB lysates. The folate calibrators, sample extraction, and chromatographic and MS conditions were as described previously (16). 5,10-Methenyltetrahydrofolic Acid hydrochloride salt (5,10CH=THF) and 10-formylfolic Acid (10CHOFA) were from Merck Eprova AG. We quantitatively isolated folates from 275 μL of serum or WB …

  • determination of folate vitamers in human serum by stable isotope dilution tandem mass spectrometry and comparison with radioassay and microbiologic assay
    Clinical Chemistry, 2004
    Co-Authors: Christine M Pfeiffer, Zia Fazili, Les Mccoy, Ming Zhang, Elaine W Gunter
    Abstract:

    Background: Current clinical methods for folate give different results and cannot measure the various forms of folate. We developed an isotope-dilution tandem mass spectrometric method coupled to liquid chromatography (LC/MS/MS) as a candidate reference method for 5-Methyltetrahydrofolic Acid (5MeTHF), 5-formyltetrahydrofolic Acid (5FoTHF), and folic Acid (FA) in human serum. Methods: We quantitatively isolated folates from 275 μL of serum with a phenyl solid-phase extraction cartridge, then detected and quantified them in stabilized serum extracts by positive-ion electrospray ionization LC/MS/MS. We used an isocratic mobile phase of acetic Acid in organic solvent on a C 8 analytical column. 13 C-labeled folates were used as internal standards. Results: Limits of detection in serum were 0.13 (5MeTHF), 0.05 (5FoTHF), and 0.07 (FA) nmol/L. Within- and between-run imprecision (CV) was 0.5 nmol/L, and 2.0 nmol/L. Total folate (TFOL) concentrations determined by competitive protein binding radioassay were ∼9% lower than results obtained with LC/MS/MS. The microbiologic assay gave ∼15% higher TFOL results with FA calibrator and no difference with 5MeTHF calibrator. The mean (SD) [range] TFOL in 42 sera was 35.5 (17.8) [6.5–75.6] nmol/L. Thirty-two samples with TFOL 50 nmol/L had, on average, 81.7% 5MeTHF, 15.7% FA, and 2.5% 5FoTHF. Conclusions: This stable-isotope-dilution LC/MS/MS method can quantify 5MeTHF, 5FoTHF, and FA in serum. Currently used clinical assays agree with this candidate reference method.

Gene E Lester - One of the best experts on this subject based on the ideXlab platform.

  • antioxidant sugar mineral and phytonutrient concentrations across edible fruit tissues of orange fleshed honeydew melon cucumis melo l
    Journal of Agricultural and Food Chemistry, 2008
    Co-Authors: Gene E Lester
    Abstract:

    Orange-fleshed, non-netted honeydew (Cucumis melo L.) is a relatively new melon in the marketplace and has shown a lot of potential as an alternative to netted muskmelons (cantaloupes), which are often prone to surface contamination by enteric bacteria. Orange-fleshed honeydew is a cross between orange-fleshed cantaloupe and non-netted, green-fleshed honeydew. This glasshouse study investigated the nutritional profile (phytonutrient and sugar contents) in different tissues of mature orange-fleshed honeydew melon fruit. The equatorial mesocarp of ripe fruit was segmented into hypodermal (subpeel), outer, middle, and inner (near the seed cavity) tissues and then assayed for total sugars, mineral nutrients, phytonutrients, total proteins, and enzymatic antioxidant activities. The concentrations of soluble solids, sucrose, total sugars, β-carotene, and 5-Methyltetrahydrofolic Acid increased in an inward direction from the subpeel mesocarp tissues toward the seed cavity. The activities of ascorbate peroxidase,...

  • antioxidant sugar mineral and phytonutrient concentrations across edible fruit tissues of orange fleshed honeydew melon cucumis melo l
    Journal of Agricultural and Food Chemistry, 2008
    Co-Authors: Gene E Lester
    Abstract:

    Orange-fleshed, non-netted honeydew ( Cucumis melo L.) is a relatively new melon in the marketplace and has shown a lot of potential as an alternative to netted muskmelons (cantaloupes), which are often prone to surface contamination by enteric bacteria. Orange-fleshed honeydew is a cross between orange-fleshed cantaloupe and non-netted, green-fleshed honeydew. This glasshouse study investigated the nutritional profile (phytonutrient and sugar contents) in different tissues of mature orange-fleshed honeydew melon fruit. The equatorial mesocarp of ripe fruit was segmented into hypodermal (subpeel), outer, middle, and inner (near the seed cavity) tissues and then assayed for total sugars, mineral nutrients, phytonutrients, total proteins, and enzymatic antioxidant activities. The concentrations of soluble solids, sucrose, total sugars, beta-carotene, and 5-Methyltetrahydrofolic Acid increased in an inward direction from the subpeel mesocarp tissues toward the seed cavity. The activities of ascorbate peroxidase, catalase, and superoxide dismutase also increased in an inward direction. The concentrations of calcium, iron, magnesium, manganese, and sodium all decreased in the inward direction. When expressed on a dry weight basis, the concentrations of ascorbic Acid, boron, copper, fructose, glucose, phosphorus, potassium, and zinc were higher in the subpeel region compared to the inner mesocarp tissues, but the reverse was true when data were expressed on a fresh weight basis. These data reveal that there is considerable variation in sugars, minerals, and phytonutrients across the mesocarp regions and that expressing the data on a fresh or dry weight basis can alter interpretations of the nutritional significance and health benefits of fruit. The data also confirm that orange-fleshed honeydew melon can be a rich source of many human health-related nutrients.

Zia Fazili - One of the best experts on this subject based on the ideXlab platform.

  • EW. Determination of folate vitamers in human serum by stable-isotope-dilution tandem mass spectrometry and comparison with radioassay and microbiologic assay. Clin Chem 2004;50:423–32
    2015
    Co-Authors: Christine M Pfeiffer, Zia Fazili, Les Mccoy, Ming Zhang, Elaine W Gunter
    Abstract:

    Background: Current clinical methods for folate give different results and cannot measure the various forms of folate. We developed an isotope-dilution tandem mass spectrometric method coupled to liquid chroma-tography (LC/MS/MS) as a candidate reference method for 5-Methyltetrahydrofolic Acid (5MeTHF), 5-formyltet-rahydrofolic Acid (5FoTHF), and folic Acid (FA) in hu-man serum. Methods: We quantitatively isolated folates from 275 L of serum with a phenyl solid-phase extraction car-tridge, then detected and quantified them in stabilized serum extracts by positive-ion electrospray ionization LC/MS/MS. We used an isocratic mobile phase of acetic Acid in organic solvent on a C8 analytical column. 13C-labeled folates were used as internal standards. Results: Limits of detection in serum were 0.13 (5MeTHF), 0.05 (5FoTHF), and 0.07 (FA) nmol/L. Within-and between-run imprecision (CV) was <7 % for 5MeTHF and <10 % for 5FoTHF at concentrations>0.5 nmol/L, and <10 % for FA at concentrations>2.0 nmol/L. Total folate (TFOL) concentrations determined by com-petitive protein binding RIA were 9 % lower than results obtained with LC/MS/MS. The microbiologic assay gave 15 % higher TFOL results with FA calibra-tor and no difference with 5MeTHF calibrator. The mean (SD) [range] TFOL in 42 sera was 35.5 (17.8) [6.5–75.6] nmol/L. Thirty-two samples with TFOL <50 nmol/L had, on average, 93.3 % 5MeTHF, 2.3 % FA, and 4.4 % 5FoTHF. Ten samples with TFOL>50 nmol/L had

  • influence of 5 10 methylenetetrahydrofolate reductase polymorphism on whole blood folate concentrations measured by lc ms ms microbiologic assay and bio rad radioassay
    Clinical Chemistry, 2008
    Co-Authors: Zia Fazili, Christine M Pfeiffer, Mindy Zhang, Ram B Jain, Deborah Koontz
    Abstract:

    Background: The 5,10-methylenetetrahydrofolate reductase (NADPH) ( MTHFR ) C677T polymorphism may affect whole-blood folate pattern measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and total folate measured by LC-MS/MS, microbiologic assay, and Bio-Rad radioassay (BR). Methods: We analyzed 171 whole blood hemolysates from 2 blood banks for folate pattern and total folate concentrations using these 3 methods and determined MTHFR genotype. Results: The median (range) total folate concentration by LC-MS/MS was higher in the US set [378 (228–820) nmol/L; n = 96] than in the European set [250 (122–582) nmol/L; n = 75]. The whole-blood folate pattern [median (range)] was similar for individuals with C/C (n = 73) and C/T (n = 66) genotype: 88% (71%–91%) and 86% (50%–91%), respectively, for 5-Methyltetrahydrofolic Acid (5CH3THF) vs 12% (9%–29%) and 14% (9%–51%) for forms other than 5-Methyltetrahydrofolic Acid (non-5CH3THF). Individuals with T/T (n = 32) genotype had 58% (22%–87%) 5CH3THF vs 42% (13%–78%) non-5CH3THF. Compared with microbiologic assay results, LC-MS/MS ( r = 0.94) and BR ( r = 0.87) results were significantly lower (−10% and −45%, respectively); however, these differences were concentration dependent and also genotype dependent for the BR assay (−48% for C/C+C/T and −31% for T/T). The microbiologic assay completely recovered [mean (SD)] folates added to a whole blood hemolysate, except for tetrahydrofolic Acid (THF) [46.4% (8.1%)]. The BR assay underrecovered 5CH3THF [51% (4.1%)] and 5-formyltetrahydrofolic Acid [18% (0.1%)], and overrecovered THF [152% (19%)]. Conclusion: MTHFR C677T polymorphism influences the folate pattern in whole blood. The agreement between total folate by LC-MS/MS and microbiologic assay, independent of the MTHFR genotype, allows the use of one regression equation. Because BR results are genotype dependent, different regression equations should be used.

  • comparison of serum folate species analyzed by lc ms ms with total folate measured by microbiologic assay and bio rad radioassay
    Clinical Chemistry, 2007
    Co-Authors: Zia Fazili, Christine M Pfeiffer, Mindy Zhang
    Abstract:

    Background: The Bio-Rad QuantaPhase II radioassay (BR), used for 25 years to measure total folate (TFOL) concentrations for the National Health and Nutrition Examination Survey (NHANES), will be discontinued in 2007. Liquid chromatography–tandem mass spectrometry (LC-MS/MS) or a microbiologic assay (MA) will be used in the future. Methods: We measured folate species by LC-MS/MS and TFOL by MA and BR in 327 serum samples. Results: LC-MS/MS measured 5-Methyltetrahydrofolic Acid (5CH3THF; 82%), folic Acid (FA; 8%), 5-formyltetrahydrofolic Acid (5CHOTHF; 6%), tetrahydrofolic Acid (THF; 4%), and 5,10-methenyltetrahydrofolic Acid (5,10CH=THF; 0%). The sum of the folate species correlated well with TFOL measured by MA ( R 2 = 0.97) and BR ( R 2 = 0.91). Compared with LC-MS/MS results, MA and BR values were significantly lower (−6% and −29%, respectively); however, these differences were concentration dependent. The MA almost completely recovered folates added to serum samples except for FA [69% (3%)] and THF [36% (10%)]. The BR underrecovered 5CH3THF [61% (9%)] and 5CHOTHF [38% (14%)] and overrecovered 5,10CH=THF [234% (32%)]. Multiple linear regression models with log-transformed data yielded a good fit for converting BR data to MA or LC-MS/MS data and MA data to LC-MS/MS data. Conclusions: The good correspondence between the sum of folate species determined by LC-MS/MS and TFOL determined by MA makes these 2 assays interchangeable. The BR produces much lower results, on average, probably because of 5CH3THF underrecovery. The conversion equations provided could be used for future NHANES time trend analyses.

  • measurement of folates in serum and conventionally prepared whole blood lysates application of an automated 96 well plate isotope dilution tandem mass spectrometry method
    Clinical Chemistry, 2004
    Co-Authors: Zia Fazili, Christine M Pfeiffer
    Abstract:

    Folate nutriture is associated with neural tube defects (1), vascular diseases (2), certain forms of cancer (3), and cognitive function (4). To reduce the risk for neural tube defects, the US Food and Drug Administration mandated folate fortification of cereal grain products beginning in January 1998 (5). Serum folate and whole blood folate (WBF) are measured to determine folate status. Clinical methods to determine serum folate and WBF, such as the microbiologic assay and various immunoassays, measure only total folate (TF) and present variable results, especially for WBF (6)(7). Few methods have described the measurement of WBF by chromatography-based methods (8)(9)(10)(11)(12)(13)(14)(15). Gas chromatography/mass spectrometry used to analyze WBF after cleavage of folates to p -aminobenzoic Acid showed greater sensitivity than previous chromatographic methods and used for the first time an isotope-labeled internal standard (IS). The method, however, requires a complex and lengthy multistep sample preparation including chemical derivatization (11)(13)(14)(15). We describe here the first automated 96-well plate stable-isotope-dilution liquid chromatography–tandem mass spectrometry (LC/MS/MS) method that measures intact folate monoglutamates in conventionally prepared whole blood (WB) lysates and serum. To increase sample throughput and minimize the extent of manual sample preparation, we adapted our recently developed manual solid-phase extraction (SPE) LC/MS/MS method for 5-Methyltetrahydrofolic Acid (5CH3THF), 5-formyltetrahydrofolic Acid (5CHOTHF), and folic Acid (FA) in serum (16) to an automated method using 96-well plates. In addition, we extended this method to measure additional folate forms in WB lysates. The folate calibrators, sample extraction, and chromatographic and MS conditions were as described previously (16). 5,10-Methenyltetrahydrofolic Acid hydrochloride salt (5,10CH=THF) and 10-formylfolic Acid (10CHOFA) were from Merck Eprova AG. We quantitatively isolated folates from 275 μL of serum or WB …

  • determination of folate vitamers in human serum by stable isotope dilution tandem mass spectrometry and comparison with radioassay and microbiologic assay
    Clinical Chemistry, 2004
    Co-Authors: Christine M Pfeiffer, Zia Fazili, Les Mccoy, Ming Zhang, Elaine W Gunter
    Abstract:

    Background: Current clinical methods for folate give different results and cannot measure the various forms of folate. We developed an isotope-dilution tandem mass spectrometric method coupled to liquid chromatography (LC/MS/MS) as a candidate reference method for 5-Methyltetrahydrofolic Acid (5MeTHF), 5-formyltetrahydrofolic Acid (5FoTHF), and folic Acid (FA) in human serum. Methods: We quantitatively isolated folates from 275 μL of serum with a phenyl solid-phase extraction cartridge, then detected and quantified them in stabilized serum extracts by positive-ion electrospray ionization LC/MS/MS. We used an isocratic mobile phase of acetic Acid in organic solvent on a C 8 analytical column. 13 C-labeled folates were used as internal standards. Results: Limits of detection in serum were 0.13 (5MeTHF), 0.05 (5FoTHF), and 0.07 (FA) nmol/L. Within- and between-run imprecision (CV) was 0.5 nmol/L, and 2.0 nmol/L. Total folate (TFOL) concentrations determined by competitive protein binding radioassay were ∼9% lower than results obtained with LC/MS/MS. The microbiologic assay gave ∼15% higher TFOL results with FA calibrator and no difference with 5MeTHF calibrator. The mean (SD) [range] TFOL in 42 sera was 35.5 (17.8) [6.5–75.6] nmol/L. Thirty-two samples with TFOL 50 nmol/L had, on average, 81.7% 5MeTHF, 15.7% FA, and 2.5% 5FoTHF. Conclusions: This stable-isotope-dilution LC/MS/MS method can quantify 5MeTHF, 5FoTHF, and FA in serum. Currently used clinical assays agree with this candidate reference method.

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  • assessing volumetric absorptive microsampling coupled with stable isotope dilution assay and liquid chromatography tandem mass spectrometry as potential diagnostic tool for whole blood 5 Methyltetrahydrofolic Acid
    Frontiers in Nutrition, 2017
    Co-Authors: Markus Kopp, Michael Rychlik
    Abstract:

    Volumetric absorptive microsamplers (VAMS) have been developed recently as a promising tool for clinical blood sampling. Compared to dried blood spot (DBS) samples analysed by accurate stable isotope dilution assays (SIDAs) the new technique could provide further substantial miniaturizing of folate assays by eliminating hematocrit effects and uneven analyte distribution within the sample. Herein we present a miniaturized SIDA coupled with LC-MS/MS measurement of 5-Methyltetrahydrofolic Acid as main folate vitamer in whole blood using [13C5]-5-Methyltetrahydrofolic Acid as internal standard. Elution and extraction of only 10.8 µL dried whole blood was carried out by centrifugation followed by enzymatic treatment for polyglutamate deconjugation. Matrix separation was achieved by heating and centrifugation. To proof applicability we screened 11 volunteers for their whole blood folate status. Limits of detection and quantitation were 9 and 26 nmol∙L-1 and, therefore, sufficiently low for screening folate status. Recoveries were 97 (±5.8), 99 (±2.8) and 96 (±6.1)% for 800, 400 and 200 nmol∙L-1 5-Methyltetrahydrofolic Acid, respectively. Precision of the LC-MS/MS instrument and inter-assay precision trials revealed CVs of 8.1 and 3.5% (294 nmol∙L-1), respectively, thus confirming reproducible and precise quantitation. Compared to fresh whole blood no significant degradation of 5-methyltetrahydrofolate was observed after 2.5 h drying at room temperature. VAMS 5-CH3-H4folate was stable for at least 3 weeks at -20°C. In our pilot study accurate and diagnostically conclusive determination of folate status was verified. Nevertheless, blood sampling should be performed by trained individuals to avoid substantial errors concerning the absorbed volume. Endogenous folate in rat serum and chicken pancreas caused a significant background especially at low blood 5-CH3-H4folate levels and, thus, removing these background folates or alternative mixtures for polyglutamate deconjugation are needed. The superior feasibility of a minimized blood collection with VAMS allows further progress regarding time- and cost-effective methodologies in newborn or population screenings for 5-methyltetrahydrofolate status. Further steps towards minimization could include an automated assay coupled with UPLC-MS/MS.

  • Measurements of Intra- and Extra-Cellular 5-Methyltetrahydrofolate Indicate that Bifidobacterium Adolescentis DSM 20083T and Bifidobacterium Pseudocatenulatum DSM 20438T Do Not Actively Excrete 5-Methyltetrahydrofolate In vitro
    Frontiers in Microbiology, 2017
    Co-Authors: Markus Kopp, Kerstin Dürr, Matthias Steigleder, Thomas Clavel, Michael Rychlik
    Abstract:

    Certain intestinal bifidobacteria have the ability to synthesize folates. In vitro experiments revealed a high production, cellular accumulation, and release of reduced folate vitamers like 5-methyltetrahydrofolate and tetrahydrofolate in folate-free medium (FFM). However, it is still unclear to which extent synthesized folates are polyglutamylated and probably not available for transport, and if they are actively released by excretion. To address these questions, we characterized intra- and extracellular pteroylmonoglutamates and polyglutamylated 5-methyltetrahydrofolate (5-CH3-H4PteGlu2-4) in B. adolescentis DSM 20083T and B. pseudocatenulatum DSM 20438T in vitro. Folates were measured by means of stable isotope dilution assays coupled with LC-MS/MS analysis using [2H4]-5-Methyltetrahydrofolic Acid, [2H4]-tetrahydrofolic Acid and [2H4]-5-formyltetrahydrofolic Acid as internal standards. Cell viability was examined by fluorescence microscopy. Quantitation of folate production by B. adolescentis during the stationary phase revealed a linear increase of dead cells paralleled by increasing concentration of 5-formyltetrahydrofolate and 5-methyltetrahydrofolate (100% 5-CH3-H4PteGlu4) in FFM, whereas the intracellular concentrations of these vitamers remained constant. After 24 h, B. adolescentis (125 mg cells, wet weight) produced a total amount of 0.846 nmol 5-CH3-H4folate: 0.385±0.059 nmol (46±7%) and 0.461±0.095 nmol (54±11%) measured in the intracellular (viable cells; 52±3% measured by fluorescence microscopy) and extracellular (lysed cells; 48±3%) fraction, respectively. For B. pseudocatenulatum (124 mg cells, wet weight), 1.135 nmol 5-CH3-H4folate was produced after 24 h, and a similar proportionality between intra/extracellular folate concentrations and viable/lysed cells was observed. These results indicate that the strains tested produce and accumulate 5-CH3-H4PteGlu4 for cellular metabolism, and that extracellular concentrations of the vitamer arise from cell lysis.

  • development of stable isotope dilution assays for the quantitation of intra and extracellular folate patterns of bifidobacterium adolescentis
    Journal of Chromatography A, 2016
    Co-Authors: Markus Kopp, Kerstin Dürr, Matthias Steigleder, Thomas Clavel, Michael Rychlik
    Abstract:

    Folate-producing bifidobacteria have been studied extensively but appropriate methods for detailed quantitation of intra- and extracellular pteroylmono- and pteroylpolyglutamate patterns are lacking. Therefore, B. adolescentis DSM 20083T was cultivated in folate-free medium (FFM) for 24h to develop and validate stable isotope dilution assays (SIDAs) coupled with LC-MS/MS for the determination of 5-formyltetrahydrofolic Acid (5-HCO-H4folate), 10-formylfolic Acid (10-HCO-PteGlu), tetrahydrofolic Acid (H4folate), folic Acid (PteGlu) and 5-Methyltetrahydrofolic Acid (5-CH3-H4folate) including its di-, tri-, and tetraglutamic vitamers (5-CH3-H4PteGlu2-4). The respective monoglutamylated isotopologues labelled with deuterium were used as internal standards for quantitation. Limits of detection and quantitation (LOD/LOQ) were sufficiently low to quantify 48.2nmol L-1 5-CH3-H4folate (5.7/17nmolL-1) and 71.0nmolL-1 5-HCO-H4folate (10/30nmolL-1) as major folate vitamers extracellularly and 124nmolL-1 5-CH3-H4folate (3.4/10nmolL-1), 213nmolL-1 5-HCO-H4folate (4.8/14nmolL-1), and 61.4nmolL-1 H4folate (2.3/7.0nmolL-1) intracellularly after deconjugation. The major portion of native 5-CH3-H4folate vitamer was ascribed to its tetraglutamate ( > 95%). Concentrations of mono-, di-, tri-, and pentaglutamylated folates were below LOD or LOQ. Intra-assay precision coefficients of variation (CVs) ranged from 7% (at a concentration of 53.9nmolL-1 for 5-CH3-H4PteGlu4), 15% (25.5nmolL-1 5-CH3-H4folate) to 18% (78.5nmolL-1 5-HCO-H4folate), extracellularly, and from 6% (60.7nmolL-1 5-CH3-H4PteGlu4), 7% (202nmolL-1 5-HCO-H4folate), 10% (67.1nmolL-1 H4folate) to 11% (127nmolL-1 5-CH3-H4folate), intracellularly. Inter-assay precision CVs ranged from 2% (54.7nmolL-1 5-CH3-H4PteGlu4), 3% (71nmolL-1 5-HCO-H4folate) to 11% (48.2nmolL-1 5-CH3-H4folate), extracellularly, and from 1% (61.4nmolL-1 H4folate), 5% (213nmolL-1 5-HCO-H4folate), 6% (63.5nmolL-1 5-CH3-H4PteGlu4) to 10% (124nmolL-1 5-CH3-H4folate), intracellularly, thus showing excellent reproducibility. Recoveries for all analytes under study ranged between 81 and 113%. These newly developed methods enable reproducible, precise and sensitive quantitation of eight bacterially synthesized folate vitamers in two totally different matrices, including both monoglutamates and polyglutamates. Furthermore, we here present the first assay using solely monoglutamylated [2H4]-5-CH3-H4folate to quantify native polyglutamate patterns of this vitamer in bacteria which might replace time-consuming determination of monoglutamates in the future.

  • stable isotope dilution assays for clinical analyses of folates and other one carbon metabolites application to folate deficiency studies
    PLOS ONE, 2016
    Co-Authors: Markus Kopp, Rosalie Morisset, Peter Koehler
    Abstract:

    : Folate deficiency is generally accepted as a potential direct or indirect risk factor for diseases including spina bifida, coronary heart diseases, malfunctions of the central nervous system, and cancer. The direct inclusion of folates in the methylation cycle, including the remethylation of homocysteine and regeneration of S-adenosylmethionine, underlines the importance of these vitamins and other components of one-carbon metabolism. Therefore, the aim of the present study was to develop a multiple stable isotope dilution assay (SIDA) for the respective analytes in plasma and tissue samples to allow for a closer look at the interaction between a severe folate deficiency and local folate status, as well as further interactions with circulating S-adenosylmethionine, S-adenosylhomocysteine, and homocysteine. The analytical methods were based on SIDAs coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis using the deuterated folates [2H4]-5-Methyltetrahydrofolic Acid, [2H4]-5-formyltetrahydrofolic Acid, [2H4]-tetrahydrofolic Acid, [2H4]-10-formylfolic Acid, and [2H4]-folic Acid and the deuterated one-carbon metabolites [2H4]-homocysteine, [2H4]-S-adenosylhomocysteine, and [2H3]-S-adenosylmethionine as internal standards. Three analytical methods have been developed for the analysis of homocysteine, S-adenosylmethionine, S-adenosylhomocysteine, and six folate vitamers. Validation data for the analysis of C1-metabolites in plasma and tissue samples or folate analysis in tissue samples revealed excellent sensitivity, precision, and recovery for all analytes studied. The miniaturized methods using sample volumes as low as 50 μL and weighed portions of 5-25 mg will allow the assessment of the status of folates and additional biomarkers of impaired one-carbon metabolism during folate deficiency.

  • Quantitation of 5-Methyltetrahydrofolic Acid in Dried Blood Spots and Dried Plasma Spots by Stable Isotope Dilution Assays.
    PloS one, 2015
    Co-Authors: Markus Kopp, Michael Rychlik
    Abstract:

    Because of minimal data available on folate analysis in dried matrix spots (DMSs), we combined the advantages of stable isotope dilution assays followed by LC-MS/MS analysis with DMS sampling to develop a reliable method for the quantitation of plasma 5-Methyltetrahydrofolic Acid in dried blood spots (DBSs) and dried plasma spots (DPSs) as well as for the quantitation of whole blood 5-Methyltetrahydrofolic Acid in DBSs. We focused on two diagnostically conclusive parameters exhibited by the plasma and whole blood 5-Methyltetrahydrofolic Acid levels that reflect both temporary and long-term folate status. The method is performed using the [2H4]-labeled isotopologue of the vitamin as the internal standard, and three steps are required for the extraction procedure. Elution of the punched out matrix spots was performed using stabilization buffer including Triton X-100 in a standardized ultrasonication treatment followed by enzymatic digestion (whole blood only) and solid-phase extraction with SAX cartridges. This method is sensitive enough to quantify 27 nmol/L whole blood 5-Methyltetrahydrofolic Acid in DBSs and 6.3 and 4.4 nmol/L plasma 5-Methyltetrahydrofolic Acid in DBSs and DPSs, respectively. The unprecedented accurate quantification of plasma 5-Methyltetrahydrofolic Acid in DBSs was achieved by thermal treatment prior to ultrasonication, inhibiting plasma conjugase activity. Mass screenings are more feasible and easier to facilitate for this method in terms of sample collection and storage compared with conventional clinical sampling for the assessment of folate status.