Assessment of the Stability of Supraphysiological Ascorbate in Human Blood: Appropriate Handling of Samples from Clinical Trials for Measurements of Pharmacological Ascorbate

Abstract

Based on encouraging results from several early-phase clinical trials, there is renewed interest in the use of pharmacological ascorbate (i.e., intravenous administration resulting in >≈10 mM plasma ascorbate concentrations) in combination with standard-of-care cancer treatments including radiation and/or chemotherapy. Under normal, healthy physiological conditions, humans maintain plasma ascorbate concentrations in the range of 40-80 lM. However, in vivo antitumor activity requires supraphysiological plasma concentrations on the order of ≈20 mM. The stability of ascorbate in whole blood has been well studied. The goal of this work was to determine the appropriate handling methods of blood samples, after treatment with pharmacological ascorbate, which allow for the optimal measurement of ascorbate in plasma for dosing verification. Our findings indicate that ascorbate concentrations (mM) are relatively stable in whole blood collected in sodium heparin tubes and stored on ice (or at 4°C) for up to 24 h. After 24 h, ascorbate levels in plasma are relatively stable at 4°C for up to 72 h. At -20°C, plasma concentrations are relatively stable for 2-3 weeks, while at -80°C, ascorbate concentrations in plasma are stable for at least one month. In contrast, patient samples showed better stability when stored as whole blood compared to plasma at 4°C but increasing hemolysis over time may significantly skew ascorbate measurements. Additionally, patient samples can be reliably stored as plasma at -20°C for up to three weeks in either a frost-containing or frost-free environment. This information can guide the collection, processing and storage of clinical samples after pharmacological ascorbate infusions amenable to multi-center clinical trials.

FIG. 1.

Ascorbate stability in whole blood. Blood samples from healthy human subjects were dosed with ascorbate (20 mM) and stored at either room temperature (RT) or 4°C. At the time of analysis, samples (n = 12) were centrifuged at 2,000g for 14 min to separate RBCs and plasma. [AscH⁻] was determined in the plasma using a microvolume UV-Vis spectrometer; values are reported as a fraction of the initial concentration ± SD. *P < 0.05 based on two-way ANOVA comparing the interaction between storage conditions.

FIG. 2.

Ascorbate stability in plasma. Blood samples from healthy human subjects were dosed with ascorbate (20 mM). Samples (n = 12) were centrifuged at 2,000g for 14 min to separate RBCs and plasma. After centrifugation, plasma samples were stored at either room temperature (RT) or 4°C. [AscH⁻] was determined in the plasma using a microvolume UV-Vis spectrometer; values are reported as a fraction of the initial concentration ± SD. * P < 0.05 based on two-way ANOVA comparing interaction between storage conditions.

FIG. 3.

Ascorbate stability in plasma at −80°C. Blood samples from healthy human subjects were dosed with ascorbate (20 mM). After addition of ascorbate, samples were centrifuged at 2,000g for 14 min to separate RBCs and plasma. Plasma samples were stored at −80°C for up to 28 days. [AscH⁻] measurements were done using a microvolume UV-Vis spectrometer at 14-day intervals for up to 28 days and are reported as a fraction of the initial concentration ± SD.

FIG. 4.

Ascorbate stability in plasma at −20°C. Blood samples from healthy human subjects were dosed with ascorbate (20 mM). After addition of ascorbate, samples were centrifuged at 2,000g for 14 min to separate RBCs and plasma. Samples (n = 12) were stored at −20°C for 5 days. [AscH⁻] measurements were done every 5–7 days for 14 days using a microvolume UV-Vis spectrometer and are reported as a fraction of the initial concentration ± SD

FIG. 5.

Ascorbate stability in patient samples stored at 4°C. Patient samples collected from phase II ascorbate lung and glioblastoma clinical trials were stored at 4°C as either plasma (n = 13) or whole blood (n = 11). Samples were centrifuged at 2,000g for 14 min to separate RBCs and plasma prior to analysis. [AscH⁻] measurements were done every 24 h for up to 96 h using a microvolume UV-Vis spectrometer and are reported as a fraction of the initial concentration.*P < 0.05 based on two-way ANOVA comparing interaction between storage and analysis conditions.

FIG. 6.

Ascorbate stability in patient samples stored at −20°C. Patient samples (n = 5) were stored as plasma for 33 days in either a frost-containing or frost-free environment, with ascorbate plasma concentrations being measured every 5–7 days. *P < 0.05 based on two-way ANOVA comparing interaction between storage conditions.