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.
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 20083 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-Hfolate), 10-formylfolic acid (10-HCO-PteGlu), tetrahydrofolic acid (Hfolate), folic acid (PteGlu) and 5-methyltetrahydrofolic acid (5-CH-Hfolate) including its di-, tri-, and tetraglutamic vitamers (5-CH-HPteGlu). 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 5-CH-Hfolate (5.7/17nmolL) and 71.0nmolL 5-HCO-Hfolate (10/30nmolL) as major folate vitamers extracellularly and 124nmolL 5-CH-Hfolate (3.4/10nmolL), 213nmolL 5-HCO-Hfolate (4.8/14nmolL), and 61.4nmolL Hfolate (2.3/7.0nmolL) intracellularly after deconjugation. The major portion of native 5-CH-Hfolate 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 for 5-CH-HPteGlu), 15% (25.5nmolL 5-CH-Hfolate) to 18% (78.5nmolL 5-HCO-Hfolate), extracellularly, and from 6% (60.7nmolL 5-CH-HPteGlu), 7% (202nmolL 5-HCO-Hfolate), 10% (67.1nmolL Hfolate) to 11% (127nmolL 5-CH-Hfolate), intracellularly. Inter-assay precision CVs ranged from 2% (54.7nmolL 5-CH-HPteGlu), 3% (71nmolL 5-HCO-Hfolate) to 11% (48.2nmolL 5-CH-Hfolate), extracellularly, and from 1% (61.4nmolL Hfolate), 5% (213nmolL 5-HCO-Hfolate), 6% (63.5nmolL 5-CH-HPteGlu) to 10% (124nmolL 5-CH-Hfolate), 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 [H]-5-CH-Hfolate to quantify native polyglutamate patterns of this vitamer in bacteria which might replace time-consuming determination of monoglutamates in the future.
Studies on one-carbon metabolism for the assessment of folate deficiency have focused on either metabolites of folate metabolism or methionine cycle. To bridge the gap between deficiency markers in these pathways we designed a dietary induced folate deficiency study using male C57BL/6N mice. After weaning (3 weeks) mice were fed a defined control diet (1 week) before being fed a folate deficient diet (n = 6 mice) and the control diet (n = 6 mice) for 12 additional weeks. Thereafter, we determined total homocysteine in plasma and folate in erythrocytes as well as S-adenosylmethionine, S-adenosylhomocysteine, and six folate vitamers in tissues including 5-methyltetrahydrofolate, 5-formyltetrahydrofolate, 5,10-methenyltetrahydrofolate, tetrahydrofolate, 10-formylfolic acid, and folic acid by means of stable isotope dilution assays coupled with liquid chromatography tandem mass spectrometry. In all organs, except heart (mainly 5-mehtyltetrahydrofolate), tetrahydrofolate constitutes the main vitamer. Moreover, in liver tetrahydrofolate was most abundant followed by 5-methyltetrahydrofolate (heart: tetrahydrofolate), 5-formyltetrahydrofolate, and 5,10-methenyltetrahydrofolate. Because of the significant decrease (p < 0.05) of folate status and S-adenosylmethionine/S-adenosylhomocysteine ratio accompanied with increasing S-adenosylhomocysteine (p < 0.05), hepatocytes are most susceptible to folate deficiency. To the best of our knowledge, we herein present the first method for simultaneous quantitation of eight metabolites for both folate and methionine cycle in one tissue sample, tHcy in plasma, and erythrocyte folate to shed light on physiological interrelations of one-carbon metabolism.
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