Does the addition of ascorbic acid to freshly prepared serum samples improve the stability of folate forms during multiple analyses and long‐term storage?

Abstract

To improve the stability of labile folate forms during repeated freeze/thawing and long‐term frozen storage of NHANES samples, 0.5% ascorbic acid (w/v) was added to freshly prepared serum samples from the 2011‐2012 survey prior to freezing them at ‐70°C. However, no ascorbic acid was added to samples collected during the first 2 months of 2011. To investigate whether the added ascorbic acid may improve the stability of the labile folate forms during repeated freeze/thawing, we compared folate data between an initial and a repeat analysis for both sample types. The loss of tetrahydrofolate (THF; least stable folate form) was large and similar in samples containing vs. not containing added ascorbic acid upon re‐analysis after either 1 wk (48% (n=51) vs. 42% (n=64)) or 1 y (84% (n=140) vs. 83% (n=127)). Other folate changes at 1 wk were small and within the expected assay variability: <5% for total folate and 5‐methyltetrahydrofolate (5‐methylTHF); 蠄0.1 nmol/L for folic acid and MeFox (an oxidation product of 5‐methylTHF) at low concentrations <1.5 nmol/L. Changes at 1 y were larger, but still acceptable: 蠄11% for total folate and 5‐methylTHF; <0.25 nmol/L for folic acid and MeFox at low concentrations <1.5 nmol/L. The Pearson correlation coefficients for all initial vs. re‐analysis results produced for the entire 2011‐2012 survey for 5‐methylTHF, folic acid, and MeFox were >0.95 regardless whether ascorbic acid was added to the sample. The addition of ascorbic acid to freshly prepared serum did not appear to improve the stability of THF during short‐term (1 wk) or long‐term (1 y) sample re‐analysis.