Laboratory analysis
Fasting blood samples collected at baseline and after 5 years were analyzed for serum ascorbic acid levels, PG I, and PG II. All samples were stored at -70 to -85°C and were analyzed simultaneously after completion of the 5-year supplementation. All assays were conducted by persons who were blinded to the intervention assignment and the questionnaire data. The serum for ascorbic acid measurement was stabilized by the addition of meta-phosphoric acid. The level of serum ascorbic acid was analyzed fluorometrically (iodine oxidation and condensation with 1,2-phenylenediamine). Serum levels of PG I and PG II were measured by radioimmunoassay in a commercial laboratory (Dinabot, SRL Co. Ltd, Tokyo).