This kit can measure GSH content in serum, plasma, cells, tissue and other samples. 

Detection principle

Reduced glutathione (GSH) is a tripeptide which composed of glutamic acid, glycine and cysteine. It is a kind of low molecular scavenger, which can remove O2-, H2O2, and LOOH. Beside as the main thiol compound of non-protein in the organization, GSH is the substrate of GSH-PX and GST which is indispensable to decomposing hydrogen peroxide for the two enzyme. What’s more, it can stabilize the enzyme containing thiol and prevent hemoglobin and other auxiliary factors from the oxidative damage. Recently, it is proved that GSH is also involved in the recovery of vitamin E to the reduction state. When lacking or depletion of GSH, it may cause producing toxic effects or increasing the toxic effects of many chemicals or environmental factors. It may be related to the increase of oxidative damage, so the amount of GSH is a vital factor to measure the body's antioxidant ability.

Detection principle

Reduced glutathione (GSH) can react with dithionitrobenzoic acid (DTNB) to produce thio-nitrobenzoic acid and glutathione disulfide. Nitromercaptobenzoic acid is a yellow compound which has the maximum absorption peak at 420 nm. The GSH content can be calculated by measuring the OD value at 420 nm.

Experimental instruments

Test tube, Micropipettor, Vortex mixer, Centrifuge, Spectrophotometer (420 nm)

Preparation of sample

1. Serum (plasma): Take 0.7 mL sample, add 0.7 mL reagent 1 and mix fully, then centrifuge at 4500 g for 10 min. Take the supernatant for test. (If the supernatant contains sediment, transfer the supernatant into a new EP tube and centrifuge again)

2. 5% tissue homogenate: Weigh the tissue accurately and add PBS (0.01M, PH7.4) at a ratio of weight (g): volume (mL) =1: 19, homogenize the tissue in ice bath, centrifuge for at 10000 g 15 min. Take 0.7 mL supernatant, add 0.7 mL reagent 1 and mix fully, then centrifuge at 4500 g for 10 min. Take the supernatant for test. Meanwhile, determine the concentration of supernatant (E-BC-K318, E-BC-K168, E-BC-K165). (Note: When absorbing supernatant, avoid a film on the surface and insert it into the supernatant to take 1 ml for determination.)

Operation steps

1. Blank tube: Add 1 mL of Reagent 1 to the 5 mL EP tube.

Standard tube: Add 1 mL of 20 μmol/L GSH standard solution to the 5 mL EP tube.

Sample tube: Add 1 mL of supernatant to the 5 mL EP tube.

2. Add 1.25 mL of Reagent 2 application solution, 0.25 mL of Reagent 3, 0.05 mL of Reagent 4 application solution to each tube.

3. Mix fully and stand for 15 min at room temperature. Set spectrophotometer to zero with distilled water and measure the OD values of each tube at 420 nm wavelength with 1 cm optical path cuvette.

Note: It can be refer to the following operating table.

Technical parameter

1. The sensitivity of the kit is 0.26 mg GSH/L.

2. The intra-CV is 1.8% and the inter-CV is 2.4%.

3. The recovery of the kit is 102%.

4. The detection range of the kit is 0.26-122.8 mg GSH/L.

Notes

1. This kit is for research use only.

2. Instructions should be followed strictly, changes of operation may result in unreliable results.

3. The validity of the kit is 6 months.

4. Do not use components from different batches of kit.

Citations