Sample collection and preparative phase

1. Sample collection and preparative phase 1) Immediately after blood collection (in heparinized tubes), mix 360 mL of blood with 40mL ofsolution 1* (see below) in 2 ml plastic tubes. 2) Shake gently for 20 s 3) Add 77 mL of 600 g/L TCA 4) Shake vigorously for 20 s 5) Centrifuge for 2 min at 12000g GSH and GSSG determination PSSG determination 1) Collect 300 mL of supernatant, add 1.7 mL of dichloromethane 2) Shake vigorously for 1 min 3) Collect 200 mL of the upper phase, add 100 mL of 1 mol/L TRIS-Cl, pH 10.8 and 300 mL 2,4-dinitrofluorobenzene (15 mL/L 4) Incubate for 2 hours at room temperature in the dark 5) Add 50 mL of 370 mL/L HCl 6) Load 20 mL onto HPLC and run as previously described (10) 1) Solubilize the pellet in a solution containing 5 g/L TCA and 1 mg/mL Na2EDTA 2) Stir until the pellet is dissolved, centrifuge at 12000g x 2 min to precipitate proteins; repeat steps 1 and 2 three times 3) Solubilize proteins in 800 mL water containing 1 mg/mL Na2EDTA, alkalinize to pH 7.8-8.0 by addition of solid NaHCO3. 4) Add 12 mL of 50 mmol/L dithiothreitol, wait for 5 min, add 80 mL TCA 600 g/L and centrifuge to discard proteins (12000g x 2 min) 5) Alkalinize 200 mL of the supernatant to pH 7.8-8.0 with solid NaHCO3 and add 15 mL monobromobimane (stock solution: 40 mmol/L dissolved in methanol). Incubate for 10 min, add 15 mL HCl 370 mL/L, load onto HPLC and run as previously described (9) Preparative and Pre-analytical phase * Solution 1: 1.5 mg/mL K3EDTA, 500 mmol/L N-ethyl-maleimide brought to pH 7.4 with solid NaHCO3