Optimized Protocol for GSH and GSSG Analysis in Blood Samples
This protocol outlines the preparative and analytical phases for determining glutathione (GSH) and glutathione disulfide (GSSG) levels in blood samples. Blood is collected in heparinized tubes and mixed with solution 1, followed by treatments with TCA and dichloromethane. The supernatant is processed through various steps including acidification and the addition of monobromobimane before HPLC analysis. The method ensures accurate quantification of these important metabolites, essential for assessing oxidative stress in physiological and pathological conditions.
Optimized Protocol for GSH and GSSG Analysis in Blood Samples
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Presentation Transcript
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