*3.2. Human Subjects and Blood Collection*

Five subjects were recruited for blood collection as part of a larger study on vitamin C metabolism approved by the Institutional Review Board for the Protection of Human Subjects at Oregon State University. Subjects were healthy non-smokers taking no prescription or non-prescription drugs that might influence vitamin C metabolism. For the study comparing anticoagulants, blood was collected after an overnight fast in vacutainers containing no additives or sodium heparin, lithium heparin, K2 EDTA, K3 EDTA, or sodium citrate (all from Becton Dickinson, Franklin Lakes, NJ, USA). Immediately after collection, aliquots of blood were collected from vacutainers and centrifuged at 16,000× *g* for 1 min for the separation of plasma from red blood cells. Aliquots of plasma were immediately acidified in 15% PCA containing 5 mM DTPA and placed at 4 °C to limit ascorbate oxidation until analysis, performed immediately, as described below.

For samples collected for the analysis of vitamin C oxidation in plasma, blood was collected in sodium heparin vacutainers from subjects after an overnight fast. Vacutainers were centrifuged in an Allegra X-15R (Beckman Coulter, Brea, CA, USA) at 4000× *g* at 4 °C for the separation of plasma. Vacutainers were then treated in one of three protocols: Oxidation control samples ("Control") were kept on ice with limited light exposure until the vacutainer was opened in a chamber previously purged with nitrogen gas. Plasma was removed and acidified in PCA with DTPA. Vacutainers prepared with the standard protocol ("Standard") were kept at room temperature and opened in ambient air before plasma samples were acidified with PCA. Vacutainers exposed to the environment ("Exposed") were opened under ambient air at room temperature and plasma placed in a clear 2 mL tube. Tubes containing plasma were then incubated at room temperature for two hours before acidification.
