TITLE: Antioxidant activity by reduced glutathione (GSH) assay: in vitro protocol
AUTHORS: Adrieli Sachett, Matheus Gallas-Lopes, Greicy M M Conterato, Ana Herrmann, Angelo Piato

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:left"><span>Considering the role of oxidative stress in the pathology of several diseases and the use of antioxidants as treatment and/or adjuvants in these conditions. Here we propose a protocol to evaluate the antioxidant capacity of compounds by the GSH method, through the ability to prevent the oxidation of GSH (reduced glutathione) induced by hydrogen peroxide (H</span><span style = "vertical-align:sub;">2</span><span style = "vertical-align:sub;vertical-align:sub;">O</span><span style = "vertical-align:sub;">2</span><span>), measured by the remaining groups of GSH that react with DTNB. This protocol was standardized at LAPCOM (Psychopharmacology and Behavior Laboratory at UFRGS) to assess biochemical parameters </span><span style = "font-style:italic;">in vitro.</span></div></div><div style = "text-align :; float : ;"><img style = "" src = "https://lh5.googleusercontent.com/sK15SDSGwqyciscBdp9BR_ro7W7nSDnnPaa7BEyTHJj0XbvnmT3Rjs0WHXyqSjxxphHyGH598LlNt_r0GcK4gTRIGNI4qGG7FCakgJbL4B6USuA62aTLOEa-CL_ERJj3gOketaqLSXE" /></div></div>

[STEPS]
?. [Preparing the reagents]
The first step is to prepare the reagents to be used in this protocol;
?. [Preparing the reagents]
Potassium phosphate buffer :    1.1.1 Weigh  of monobasic potassium phosphate (KH2PO4) in a beaker of appropriate size;    1.1.2 Dissolve the salt with  of ultrapure water;    1.1.3 Transfer the solution to a  volumetric flask;    1.1.4 Using ultrapure water, complete the solution's volume to reach ;    1.1.5 Weigh  of dibasic potassium phosphate (K2HPO4) in a beaker of appropriate size;    1.1.6 Dissolve the salt with  of ultrapure water;    1.1.7 Transfer the solution to a  volumetric flask;    1.1.8 Mix both solutions slowly in a  beaker following the steps below;Transfer  of the monobasic potassium phosphate (KH2PO4) solution to the beaker;Use a pH sensor to evaluate your solution. Expected conditions:;            If the pH of your solution is lower than 6.8 adjust the pH adding drops of the dibasic potassium phosphate (K2HPO4) solution;            If the pH of your solution is above 6.8 adjust the pH adding drops of the monobasic potassium phosphate (KH2PO4) solution;After adjusting the pH of this initial solution, proceed to add, slowly, drops of both buffer solutions (monobasic potassium phosphate and dibasic potassium phosphate). Use Pasteur pipettes to add the solutions. Mix your solutions using a pH sensor, making sure the mix of both buffers is always at ;
13.609 g
90 mL
100 mL
100 mL
17.418 g
90 mL
100 mL
500 mL
50 mL
?. [Preparing the reagents]
Reduced glutathione (GSH) :    1.2.1 Weigh  of GSH;    1.2.2 Dissolve completely the GSH in  of ultrapure water using a container of appropriate size;    1.2.3 Stock this solution at , in samples of  using plastic microtubes;    1.2.4 On the day of the in vitro test, remove one of the samples from the freezer;    1.2.5 Using a micropipette, transfer the content of the microtube () to a test tube;    1.2.6 Wash the microtube used to store your sample one time with  of ultrapure water and transfer this volume to the test tube;    1.2.7 Add  of ultrapure water to the test tube to obtain a solution of GSH ;
0.02028 g
1.1 mL
-20 °C
220
220
980
1000
?. [Preparing the reagents]
5,5′-Dithiobis(2-nitrobenzoic acid) (DTNB) :    1.3.1 Weigh carefully  of DTNB in a piece of aluminum foil;    1.3.2 Transfer the DTNB to a beaker of appropriate size;    1.3.3 Add  of absolute ethanol to the beaker to dissolve the salt;    1.3.4 Transfer your solution to a  volumetric flask;    1.3.5 Using absolute ethanol, complete the solution's volume to reach ;    1.3.6 Store the solution in an amber flask of appropriate size covered with aluminum foil at ;    1.3.7 On the day of the in vitro test, remove the solution from the fridge;    1.3.8 Using a micropipette, transfer  of the solution to a  volumetric flask;    1.3.9  Using ultrapure water, complete the solution's volume to reach . You will obtain a solution of DTNB ;
0.0396 g
9 mL
10 mL
10 mL
8 °C
1.25 mL
100 mL
100 mL
?. [Preparing the reagents]
Hydrogen peroxide (H2O2) solution: Follow the steps described in section 2 of this protocol to first standardize and then prepare a hydrogen peroxide solution. This reagent should be prepared on the day of the biochemical assay, it should not be stored for later use;
?. [Standardization of H202 solution]
Every 30 days, or whenever a new bottle of hydrogen peroxide is opened, the absorbance of the concentrated solution should be checked.
?. [Standardization of H202 solution]
First, we must calculate the molarity of the H2O2 solution in the bottle:Concentration stated in the bottle:  Molecular weight: 34.0147 g/mol1 M ------------ 1 mol ---------- 1000 mL 1 M ----- -----34.0147 g ------- 1000 mLx -------------- 350 g ---------1000 mL x =
?. [Standardization of H202 solution]
Prepare a  solution () of H2O2:    2.2.1 Follow the calculations below to determine the volume of the primary solution needed:C1 x V1 = C2 x V2 x V1 =  x V1 =     2.2.2 Using a micropipette, collect  of H2O2 and transfer to a  volumetric flask;    2.2.3 Using ultrapure water, complete the solution's volume to reach ;
10 mL
10 mL
9.72
9.72 µl
10 mL
10 mL
?. [Standardization of H202 solution]
Read the absorbance of the solution at  in a microplate reader;
240
?. [Standardization of H202 solution]
Calculate the real concentration of your sample following these steps:A standardized  H2O2 solution has an absorbance of 0.39410 mM H2O2 ---------------- 0.394 Abs                                          X          ---------     Absorbance of your sampleX = Represents the real concentration of the hydrogen peroxide sample
?. [Standardization of H202 solution]
Calculate the real concentration of the bottled solution following these steps:As X (calculated on the last step) Represents the real concentration of the hydrogen peroxide sample10 mM H2O2 ---------------- 10.29 M     X         ---------     ZZ = Real concentration of hydrogen peroxide in the bottle
?. [Standardization of H202 solution]
Prepare a  stock solution of H2O2: this reagent should be prepared on the day of the biochemical assay, it should not be stored for later use;    2.6.1 Follow the calculations below to determine the volume of the primary solution needed:C1 x V1 = C2 x V2Z (the real concentration determined on the last step) x V1 =  x V1 = depends on the concentration of your hydrogen peroxide solution    2.6.2 Using a micropipette, collect the determined volume of H2O2 and transfer it to a  volumetric flask;    2.6.3 Using ultrapure water, complete the solution's volume to reach ;    2.6.4 Store this solution in an amber flask at  when needed;
10 mL
10 mL
10 mL
8 °C
?. [Standardization of H202 solution]
Prepare a  solution from your  stock solution:    2.7.1 Follow the calculations below to determine the volume of the stock solution needed:C1 x V1 = C2 x V2  x V1 =  x V1 =     2.6.2 Using a micropipette, collect  of the stock solution and transfer it to a  volumetric flask;    2.6.3 Using ultrapure water, complete the solution's volume to reach ;    2.6.4 Store this solution in an amber flask at  when needed;
10 mL
333.33
333.33
10 mL
10 mL
8 °C
?. [Incubation of the samples]
To optimize the reaction, an incubation step is needed.
?. [Incubation of the samples]
Prepare  microtubes, to be used to store the samples, with the correct information. Wrap the microtubes in aluminum foil. The number of microtubes depends on the number of samples. You should provide at least five replicates (n = 5) of each sample with at least one control tube per sample. You should also prepare negative and positive control samples for your GSH solution. Perform the test at least two times to ensure the results are as correct as possible.
1.5 mL
?. [Incubation of the samples]
Incubate all your samples in the dark at  for ;
25 °C
?. [Reading your samples]
Prepare to read the absorbance of your samples in a microplate reader;
?. [Reading your samples]
Use a conventional 96-well microplate to run your samples. Before start pipetting, each well of the microplate should be marked for sample identification.
?. [Reading your samples]
Add  of DTNB to all of the wells needed. Fill two additional wells with  of DTNB to work as the negative control for the absorbance reading;
235
250
?. [Reading your samples]
Transfer  of the content of each microtube to its corresponding well in the microplate;
15
?. [Reading your samples]
Let your samples rest for ;
?. [Reading your samples]
Read the absorbance of the samples at  in a microplate reader;
412
?. [Calculating data and determinig results]
Prepare to analyze the results obtained after reading the absorbance of the samples;
?. Determine the percentage of inhibition of the hydroxyl radical: % of inhibition of the hydroxyl radical =
?. [Calculating data and determinig results]
Subtract the absorbance of the blank from the absorbance of the samples:Sample absorbance = Sample - Sample blank
?. [Calculating data and determinig results]
Determine the percentage of remaining sulfhydryls of GSH:
?. [Calculating data and determinig results]
Results should be expressed as % of remaining sulfhydryls of GSH.
?. [Calculating data and determinig results]
Calculate the mean absorbance of your replicates;
?. [Incubation of the samples]
For each sample, fill the plastic microtubes as described below. Using a micropipette fill the tubes in this order: Potassium phosphate buffer + H2O2 + GSH + Sample. Mix the solution with the pipette tip to homogenize the content; MicrotubesBuffer (µL)GSH (µL) Sample (µL) H2O2 (µL) Control blank210--40Sample blank200-1040Control17040-40Sample160401040
MicrotubesBuffer (µL)GSH (µL) Sample (µL) H2O2 (µL) Control blank210--40Sample blank200-1040Control17040-40Sample160401040