Folate Forms in Red Blood Cell Lysates and in Intact Washed Red Blood Cells Are Stable for a Few Days During Refrigerated Storage

Abstract

Abstract Objectives We investigated whether folate forms are stable in washed red blood cells (RBC) stored refrigerated for up to 9 days relative to RBC-lysates in ascorbic acid under the same storage conditions. Methods We prepared washed RBCs from freshly collected EDTA blood (n = 6 donors). For RBC-lysate 1, we diluted RBCs with saline (1/2 dilution), mixed well and prepared lysates (1/11 dilution) with 1% ascorbic acid (2 vials/time point; n = 12 vials/donor) for storage at 4°C for ≤ 9 days (baseline, 1, 2, 3, 6, and 9). For RBC-lysate 2, we aliquoted ∼0.5 mL washed RBCs (1 vial/time point; n = 5 vials/donor) for storage at 4°C for ≤9 days (1, 2, 3, 6, and 9). When refrigerated storage of samples was completed, RBC lysate 1 samples were frozen at −70°C, while the washed RBCs were diluted with saline (1/2 dilution), mixed well and RBC-lysates 2 were prepared (1/11 dilution) with 1% ascorbic acid (2 vials/time point; n = 10 vials/donor). All samples were stored at −70°C until analysis. At the time of analysis, we processed samples (2 replicates/time point; n = 2 days) for folate polyglutamate deconjugation using recombinant exo γ-glutamyl hydrolase, and conducted sample clean-up by automated solid phase extraction prior to analysis by LC-MS/MS. Results We found negligible losses of major folate forms after overnight refrigerated storage, and folate losses gradually increased over time (∼5% by day 6). The loss of 5-methyltetrahydrofolate and total folate (mean ± SD) after 2 days of storage for RBC lysate 1 was 1.2% ± 1.5% and 1.0% ± 1.3%, and for RBC-lysate 2 was 2.2% ± 1.1% and 2.7% ± 1.9%, respectively. The baseline concentration of non-methyl folate (sum of minor folate forms: 5-formyltetrahydrofolate [<LOD], tetrahydrofolate, and 5, 10-methenyltetrahydrofolate) was small (<3.0 nmol/L) in both sample types, and we noticed a slight increase (∼10%) after overnight storage. 5,10-Methenyltetrahydrofolate appeared to gradually convert to tetrahydrofolate upon storage in RBC-lysate 2 samples. We observed no noticeable changes for MeFox in either sample type. Conclusions Overnight refrigerated storage of RBC-lysates or washed RBCs is feasible and avoids notable losses of folate forms. Prolonged refrigerated storage promotes interconversions of minor folate forms. Funding Sources This work was supported by direct appropriations from U.S. Congress.