Addition of Exogenous γ-Glutamyl Hydrolase Eliminates the Need for Lengthy Incubation of Whole-Blood Lysate for Quantitation of Folate Vitamers by High-Performance Liquid Chromatography-Tandem Mass Spectrometry

Abstract

Background: Measurement of folate monoglutamates by HPLC-tandem mass spectrometry (HPLC-MS/MS) in whole-blood lysate (WBL) requires lengthy incubation before analysis, risking degradation of labile folate vitamers.

Objective: We explored whether the addition of a commercially available recombinant exogenous γ-glutamyl hydrolase (exoGGH) enzyme reduced the required incubation time of WBL for measurement of folate as monoglutamates.

Methods: For conventional deglutamylation of polyglutamates, WBL was incubated for 4 h at 37°C. Alternatively, we added exoGGH to WBL at varying concentrations (1-10 µg/mL) and incubation times (0-90 min). We also investigated modifications to the sample diluent (pH, ascorbic acid compared with sodium ascorbate, and ascorbate concentration). Finally, we tested the effect of the enzyme in different sample types: WBL from frozen whole blood compared with frozen WBL or with frozen washed RBCs. Samples (n ≤ 15/experiment) were analyzed by HPLC-MS/MS for 6 folate monoglutamates and 5-methyltetrahydrofolate diglutamate.

Results: Optimal deconjugation of folate polyglutamates was achieved by using 1% ascorbic acid and 5 µg enzyme/mL WBL, requiring ≤30 min incubation time to achieve complete folate recovery as monoglutamates. This treatment resulted in similar folate concentrations as conventional deglutamylation (4 h at 37°C). The exoGGH enzyme was effective in samples stored frozen as whole blood and as WBL. However, the extended thaw time of whole blood resulted in 5-methyltetrahydrofolate loss and unacceptable changes to the non-methyl folate concentration. Total folate (with exoGGH) measured in washed RBCs was ∼15% lower than RBC folate calculated from WBL concentrations (conventional deglutamylation).

Conclusions: The use of exoGGH minimized incubation time and thus may avoid degradative losses of labile folate forms during sample preparation. The lower folate results in washed RBCs may be due to inadequate packing of RBCs, among other unidentified factors. A larger study is required to confirm the lack of differences in folate concentrations determined with and without the use of exoGGH.

Keywords: RBC folate; deconjugation; diglutamate; human recombinant protein; monoglutamate; washed erythrocytes.

FIGURE 1

Schematic diagram of the experimental procedures. AA, ascorbic acid; exoGGH, exogenously added γ-glutamyl hydrolase; HCT, hematocrit; HPLC-MS/MS, HPLC–tandem MS; NaAsc, sodium ascorbate; RT, room temperature; SPE, solid-phase extraction; WB, whole blood; WBL, whole blood lysate.

FIGURE 2

Effect of extended thaw time on thawed whole-blood (represented by dotted lines) and whole-blood lysate (represented by solid lines) concentrations of 5-methylTHF, MeFox, and non-methyl folate. Values are means ± SDs from enzyme-treated samples (5 µg/mL); n = 8 samples with low non-methyl folate concentrations (A, C, E) and n = 7 samples with high non-methyl folate concentrations (B, D, F). Non-methyl folate is the sum of 5-formyltetrahydrofolate, tetrahydrofolate, and 5,10-methenyltetrahydrofolate. *Difference between thawed whole blood and thawed whole blood lysate, P < 0.05. MeFox, pyrazino-s-triazine derivative of 4α-hydroxy-5-methylTHF; 5-methylTHF, 5-methyltetrahydrofolate.