Fig. 1 Mechanism of total glutathione quantification

Fig. 2 Absorption spectrum of 5-Mercapto-2-nitrobenzoic acid in phosphate buffer (pH 7.5)

Assay Procedure

Tissue (100 mg)1. Homogenize the tissue in 0.5-1.0 ml 5% SSA.2. Centrifuge the homogenized tissue sample at 8,000 g for 10 min.3. Transfer the supernatant to a new tube and add ddH₂O to reduce the concentration of SSA from 0.5 to 1%. Use it for the assay.

Plasma1. Centrifuge anticoagulant-treated blood at 1,000 g for 10 min at 4°C.2. Transfer the top plasma layer to a new tube and add 5% SSA equivalent to half of the volume of the plasma.3. Centrifuge at 8,000 g for 10 min at 4°C4. Transfer the supernatant to a new tube, and add ddH₂O to reduce the concentration of SSA from 0.5 to 1%. Use it for the assay.

Erythrocytes1. Centrifuge anticoagulant-treated blood at 1,000 g for 10 min at 4°C.2. Discard the supernatant and the white buffy layer.3. Lyse the erythrocytes with 5% SSA equivalent to 4 times the volume of the erythrocytes.4. Centrifuge at 8,000 g for 10 min at 4°C.5. Transfer the supernatant to a new tube, and add ddH₂O to reduce the concentration of SSA from 0.5 to 1%. Use it for the assay. Erythrocytes can be isolated from the remaining sample solution after the plasma sample isolation.

Preparation of Assay SolutionsPreparation of 5% 5-Sulfosalicylic Acid (SSA) SolutionNote: SSA is not included in this kit.1. Dissolve 1 g SSA in 19 ml water.2. Store the solution at 4°C (stable for 6 months at 4°C).

Substrate Working SolutionAdd 1 ml Buffer Solution to 1 vial of Substrate, and dissolve. Substrate working solution is stable for 2 months at -20ºC.Enzyme Working SolutionMix Enzyme solution with pipetting before using. Take out 20 μl Enzyme solution and mix it with 4 ml Buffer solution. Enzyme working solution is stable for 2 months at 4ºC.Coenzyme Working SolutionAdd 0.7 ml ddH₂O to the Coenzyme vial and dissolve. If you don’t use all of the coenzyme working solution in one day, aliquot it into microtubes and store at -20ºC. If you use all of the coenzyme working solution in one day, just add 6.3 ml Buffer solution to the vial.The Coenzyme vial is under vacuum pressure; carefully open the cap or use a syringe to add Buffer solution. Since the Coenzyme working solution dissolved in the Buffer solution is not stable, use it in one day. The coenzyme solution prepared with ddH₂O is only stable for 2 months at -20^oC. Dilute 10 times with Buffer solution to prepare Working solution prior to use.GSH Standard SolutionsTo prepare 200 μM GSH standard solution, add 2 ml of 0.5-1% SSA to the Standard GSH vial and dissolve. Dilute 100 μl of the 200 μM GSH standard solution with 100 μl of 0.5% SSA, and repeat using serial dilution to prepare the following GSH standard solutions:100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.13 μM, 1.56 μM and 0.The Standard GSH vial is under vacuum pressure; carefully open the cap or use a syringe to add SSA. GSH powder is difficult to see. The GSH standard solutions are stable for 2 months at -20°C.

Total Glutathione Detection – Standard MethodDetection Range: 5-100 μM1. To each well, add 20 μl of Enzyme working solution, 140 μl of Coenzyme working solution, and 20 μl of either one of the GSH standard solutions or the sample solution.a)2. Incubate the plate at 37°C for 10 min.3. Add 20 μl of Substrate working solution, and incubate the plate at 37°C for 5-10 min.4. Read the absorbance at 405 nm or 415 nm using a microplate reader.5. Determine the concentration of GSH in the sample solution using a calibration curveb).

^a) Adjust the concentration of SSA in the sample solution to 0.5-1% with ddH₂O before the assay. High concentrations of SSA (>1 %) interfere with the assay.^b) Since the colorimetric reaction is stable and the O.D. increases linearly over 30 min, GSH concentration can be determined by kinetic or pseudo-endpoint (no stopping reaction, quick measurement of O.D. at certain time periods between 5 and 10 min) methods.

Total Glutathione Detection – High Sensitivity Method Detection Range: 0.5-25 μM1. To each well, add 20 μl of Enzyme working solution, 140 μl of Coenzyme working solution, and 20 μl of either one of the GSH standard solutionsa) or the sample solutionb).2. Incubate the plate at 30°C for 10 min.3. Add 20 μl of Substrate working solution, and incubate the plate at 37°C for 20-40 min.4. Read the absorbance at 405 nm or 415 nm using a microplate reader.5. Determine the concentration of GSH in the sample solution using a calibration curve.

^a) Prepare 50 mM GSH standard solution, and then prepare different concentrations of GSH standard solutions by serial dilution with 0.5% SSA as follows: 25 μM, 12.5 μM, 6.25 μM, 3.13 μM, 1.56 μM, 0.78 μM, 0.39 μM and 0.^b) Adjust the concentration of SSA in the sample solution to 0.5-1% with ddH₂O before the assay. Higher concentrations of SSA (>1%) interfere with the assay.

Determination of Total Glutathione (GSH and GSSG) ConcentrationDetermine the total glutathione concentration in the sample solution using the following equations. Since the values obtained by these equations are the amount of total glutathione in treated sample solutions, further calculations are necessary if the actual concentration of glutathione in cells or tissues needs to be determined.

Calibration Curve

Fig. 4 Calibration curves prepared using pseudo-endpoint method and kinetic method^A) Calibration curve prepared using pseudo-endpoint method. 10 min incubation at room temperature.^B) Calibration curve prepared using kinetic method.

Pseudo-endpoint method:Total glutathione = (O.D._sample O.D._blank) / slope^a)Kinetic method:Total glutathione = (Slope_sample^b)-Slope_blank^b))/slope^b)

^a)The slope of the calibration curve prepared by the pseudo-endpoint or kinetic method.^b)The slope of the kinetic reaction.