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Assay of Vitamin C in Red Blood Cells

Although vitamin C (ascorbate) is present in whole blood, accurate measurements in red blood cells (RBCs) are problematic because of assay interferences, limited sensitivity, and sample volume requirements. We describe a new technique for ascorbate measurement in RBCs of humans, wild type mice, and mice unable to synthesize ascorbate. Exogenously added ascorbate was fully recovered even when endogenous RBC ascorbate was below detection threshold (25 nM). Twenty Âμl of whole blood or 10 Âμl of packed RBCs were sufficient for assay. RBC ascorbate was stable for 24 hours from whole blood samples at 4°C. Processed, stored samples were stable for > one month at −80°C. Unlike other tissues, ascorbate concentrations in human and mouse RBCs were linear in relation to plasma concentrations (R = 0.8, 0.9 respectively). In healthy humans, RBC ascorbate concentrations were 9–57 ÂμM, corresponding to ascorbate plasma concentrations of 15–90 ÂμM. Mouse data were similar. In human blood stored as if for transfusion, initial RBC ascorbate concentrations varied approximately 6 fold, and decreased 50% after 6 weeks of storage under clinical conditions. With this assay, it becomes possible for the first time to characterize ascorbate function in relation to endogenous concentrations in normal RBCs and those stored for transfusion.