2.7.1. Ascorbate and Dehydroascorbate

The contents of ascorbate and dehydroascorbate (DHA) were determined following the procedures of Stevens et al. [28]. Three grams of frozen sample were homogenized in 12 mL of cold 6% TCA. The extract was passed through two layers of Miracloth (Calbiochem, San Diego, CA, USA). Two assays were performed on each sample to analyze the total ascorbate and reduced ascorbate. The first assay measured the total ascorbate (incubation with 5 mM dithiothreitol (DTT) for reduction of the oxidized ascorbate), and another assay measured the reduced ascorbate (without DTT). The content of DHA was determined from the difference between the total and the reduced ascorbate content. In each sample, after the filtrate (1 mL) was mixed with 1 mL of 0.4 M phosphate buffer (pH 7.4) with 5 mM DTT or 0.4 M phosphate buffer (pH 7.4), it was incubated at 37 ◦C for 20 min. After incubation, the reaction solution was added with 5 mL of 0.5% *N*-ethyl malemide for the total ascorbate assay, or 5 mL of 0.4 M phosphate buffer (pH 7.4) for the reduced ascorbate assay, and left for 1 min at room temperature, and finally was added with 4 mL of color reagent. The absorbance was determined spectrophotometrically at 550 nm (Jasco V-530, Jasco, Tokyo, Japan) after 40 min incubation at 37 ◦C. The AsA content was calculated from a standard curve. The color reagent was as follows: solution A comprised of 31% orthophosphoric acid, 4.6% TCA, and 0.6% iron chloride; and solution B, which included 4% 2,2-dipyridyl (made up of 70% ethanol). Solutions A and B were mixed 2.75 parts (A) to 1 part (B). The content of AsA was estimated by a standard curve.
