Determination of 2,3-DPG

To prepare samples for 2,3-DPG measurement, 2 ml of venous blood in heparinized tubes was collected, placed immediately on ice, deproteinized with 0.6 M perchloric acid (Sigma-Aldrich, Saint Louis, MO, United States) to lyse RBC, and neutralized with 2.5 M potassium carbonate (Sigma-Aldrich, Saint Louis, MO, United States). The supernatant was kept for at least 60 min in an ice bath and centrifuged at 3,000 × g for 10 min. The supernatant was stored at 28°C, and 2,3-DPG levels were measured using the Roche diagnostic kit (no. 10148334001). The Roche 2,3-DPG assay is based on enzymatic cleavage of 2,3-DPG, and oxidation of nicotinamide adenine dinucleotide recorded by spectrophotometry. The 2,3-DPG assays were performed in three batches and in the range of 0.02–0.15 μmol. Concentration of 2,3-DPG was calculated according to the procedure proposed by the manufacturer. The 2,3-DPG levels were normalized to the corresponding hematocrit value from the same sample. Since the concentration of 2,3-DPG rapidly decreases during storage (Hamasaki and Yamamoto, 2000; Llohn et al., 2005), the procedure for determining the 2,3-DPG level was performed immediately after taking the blood samples. Determination of 2,3-DPG level was carried out in duplicate on each sample. The reliability of 2,3-DPG measurement was evaluated based on the coefficient of variation (CV) using the test–retest method (Atkinson and Nevill, 1998). CV for 2,3-DPG was between 0.30 and 0.76%, which indicates that these measurements are characterized by a high degree of reliability.