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.
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 extra-cellular pteroylmonoglutamates and polyglutamylated 5-methyltetrahydrofolate (5-CH3-H4PteGlu2−4) in Bifidobacterium adolescentis DSM 20083T and Bifidobacterium pseudocatenulatum DSM 20438T in vitro. Folates were measured by means of stable isotope dilution assays (SIDA) 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- and extra-cellular 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.
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