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Modifications to Ascorbate Concentration and pH Result in Faster Deconjugation of Whole Blood Folate Polyglutamates to Monoglutamates for Measurement by Liquid Chromatography‐Tandem Mass Spectrometry
Author(s) -
Stamm Rosemary A,
Fazili Zia,
Pfeiffer Christine M
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.802.23
Subject(s) - chemistry , chromatography , ascorbic acid , incubation , dilution , tandem mass spectrometry , liquid chromatography–mass spectrometry , mass spectrometry , biochemistry , food science , physics , thermodynamics
Objective Folate in red blood cells is polyglutamylated and requires deconjugation of glutamate residues by the enzyme γ glutamyl hydrolase (GGH) (E.C. 3.4.19.9) during an incubation step prior to analysis. The reported pH optimum for human GGH is 4.5. A lengthy incubation time is required for measurement of whole blood total folate by liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) to ensure all folates are measured as folate monoglutamates. We aimed to reduce the time taken for enzymatic cleavage of folate polyglutamates to monoglutamates. Methods We investigated modification of pH and ascorbate concentration to the hemolysate diluent to optimize deconjugation of folate polyglutamates. Preliminary testing was performed on four samples, two with high non‐methyl folate concentrations. Results The conventional dilution of whole blood (1/11 in 1% ascorbic acid solution, pH 2.7) resulted in a mean hemolysate pH of 3.8. Dilution with 0.5% ascorbic acid (pH 2.9) resulted in a mean hemolysate pH of 4.2 and dilution with 1% ascorbic acid adjusted to pH 4.1 resulted in a mean hemolysate pH of 4.4. Both conditions resulted in <1% of 5 methyltetrahydrofolate (5‐methylTHF) remaining as dilglutamate after 3 h incubation at 37°C, while ~2% of 5 methylTHF remained as diglutamate after 4 h incubation in conventionally prepared hemolysates. However, both conditions also resulted in a greater loss of tetrahydrofolate (THF) internal standard compared with the conventional dilution, likely resulting in a corresponding greater overestimation of THF concentration. In a follow up experiment, 2% sodium ascorbate solution offered better protection of THF internal standard compared with 1% sodium ascorbate (both adjusted to achieve a hemolysate pH of 4.3). However, 2% sodium ascorbate resulted in inadequate deconjugation with >4% of 5 methylTHF remaining as diglutamate after 4 h incubation, while <1% remained as 5‐methylTHF diglutamate in 1% sodium ascorbate. Conclusion The conventional dilution of whole blood results in a pH that is more acidic than the pH optimum of human GGH. Faster and more complete deconjugation of folate polyglutamates can be achieved either by reducing the ascorbic acid concentration or increasing the diluent pH to achieve a hemolysate pH closer to 4.5. Increasing the ascorbate concentration provides additional protection of THF, however, results in slower and less complete deconjugation, thereby resulting in under‐recovery of total folate. An additional means of protecting THF during incubation is required to accurately measure whole blood hemolysate total folate by LC‐MS/MS.