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How to Use GSH Levels in Detecting ROS

Glutathione, or GSH, is a non-essential tripeptide made from cysteine, glutamate and glycine. It is a powerful antioxidant that protects the cells and organs of your body from the harmful effects of ROS, or reactive oxygen species, that are formed from molecular oxygen as a result of various metabolic processes in the body. Antioxidants, such as glutathione, neutralize the ROS and prevent them from interacting with and damaging the DNA and protein in your cells. The ratio of oxidized and reduced form of GSH in your body is indicative of the ROS-related oxidative stress in your body.

Things You'll Need

Syringe
Butterfly needle
Centrifuge
Sodium heparin
Freezer
Sulfosalicylic acid
GSH Reaction Agent
Microplate
Light source
GSH standard solution

Instructions

- 1
  Draw approximately 2 ml of blood by venipuncture to the anticubetal vein with a 23-gauge butterfly needle attached to a 5 ml syringe. Add the sample to test tubes containing sodium heparin and gently invert the tube for mixing.
- 2
  Collect the red blood cells by centrifuging the sample at 2,500 revolutions per minute for five minutes. Freeze and thaw the sample three times to break the cells. Centrifuge the cell lysate solution for 10 minutes at 12,000 rpm and 4 degrees, and collect the supernatant.
- 3
  Remove the protein from the sample by adding 5 percent sulfosalicylic acid and centrifuging the sample at 12,000 rpm for five minutes at 4 degrees. Store the deproteinated supernatant at -112 degrees.
- 4
  Mix a thiol detection agent, GSH reductase, nicotinamide adenine dinucleotide phosphate and assay buffer in proportions indicated by the kit manufacturer to make the GSH reaction mixture. The amount of each chemical depends upon the number of tests you intend to perform. Dilute the GSH reaction mixture in 5 percent sulfosalicylic acid to get concentrations of 16, 32, 63, 125, 250 and 500 microliters.
- 5
  Add 1 to 10 microliters of the deproteinated blood sample into the wells of a microplate. Add serially diluted GSH standard solution, which is available in the testing kit, to each well to bring the total volume of each well to 10 microliters.
- 6
  Add 90 microliters of GSH reaction mixture that you made in Step 4 to each well in the microplate. Mix the reagent by shaking the plate for 30 seconds.
- 7
  Measure the absorbance immediately by placing the plate under light with wavelengths of 405 and 415 nanometers. Incubate the plate for 30 minutes. Take absorbance readings after every five minutes during the incubation.
- 8
  Dilute 6 millimolar of the processed sample with 5 percent sulfosalicylic acid and trifluromethane sulfonate solution. This will remove all the reduced GSH from the solution. Add 1 to 10 micoliters of this solution to the wells of microplate and repeat steps 5, 6 and 7. This will help estimate the amount of oxidized GSH in the sample.
- 9
  Plot the absorbance and sample concentrations on a graph to obtain the GSH curve for both reduced and oxidized glutathione. Calculate the ratio between both forms of GSH. Increased levels oxidative GSH levels indicate oxidative stress and ROS.

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