Development of stable isotope dilution assays for the quantitation of intra- and extracellular folate patterns of Bifidobacterium adolescentis

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 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.

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

“…As the stationary phase is characterized by a constant number of viable cells and an increasing number of dead cells, folate content should increase in the extracellular fraction and remain approximately constant in cells according to our hypothesis. As depicted in Figures 1C–F, we measured the three main monoglutamylated folate vitamers and 5-CH 3 -H 4 PteGlu 4 , identified as the predominant polyglutamylated 5-CH 3 -H 4 folate in both cells and FFM (Kopp et al, 2016), between 15 and 45 h of incubation. The further vitamer 5-HCO-H 4 folate (Figure 1F) showed curve shape and concentrations similar to that of 5-CH 3 -H 4 folate.…”

“…All chemicals used for cultivation of bifidobacteria, reagents for extraction, and LC-MS/MS solvents have been published earlier (Kopp et al, 2016). Rat serum (preservative free) was obtained from Biozol (Eching, Germany).…”

“…Aliquot 1 was subjected to fluorescence microscopy (Section Determination of Relative Viability by Fluorescence Microscopy), aliquot 2 was resuspended in 4 mL PBS, centrifuged for 15 min at 5000 g and 4°C. The supernatant was discarded and cellular fraction was resuspended in 10 mL extraction buffer and subjected to monoglutamate or polyglutamate extraction as described recently (Kopp et al, 2016). …”

“…Extra- and intra-cellular pteroylmono- and pteroylpoly-glutamates were determined separately by means of LC-MS/MS according to our method described previously (Kopp et al, 2016). …”

“…We recently developed a stable isotope dilution assay (SIDA) coupled with LC-MS/MS for sensitive quantitation of the main folate vitamers accumulated and released by Bifidobacterium adolescentis DSM 20083 T , including 5-CH 3 -H 4 folate, 5-HCO-H 4 folate, H 4 folate, and 5-CH 3 -H 4 PteGlu 2−4 (Kopp et al, 2016). Moreover, we found that the major portion of native 5-CH 3 -H 4 folate vitamer was present as its tetraglutamate (>95%) intra- and extra-cellularly.…”