Measurement of Oxidized/Reduced Glutathione Ratio - Protein Misfolding and Cellular Stress in Disease and Aging - page 264

7. Once a concentration of GSSG is obtained, subtract the
GSSG level value from the total GSH concentration obtained
in Subheading 3.6 from the corresponding sample to obtain
the value for reduced GSH.
8. Use the following equation to obtain the GSH/GSSG ratio:

GSH Reduced GSH (Section 3.4 result − Section 3.5 result)

=

GSSG GSSG (Section 3.5 result)

4. Notes

1. All the buffers and reagents described in Subheadings 2.2 and
2.3 can be purchased in a kit for easy preparation from
Caymen Chemical.
2. Only the total GSH content should be measured in plasma
using this assay. The concentration of GSH in plasma is below
the detection limit of this assay. To circumvent this issue, plasma
samples can be lyophilized and reconstituted with MES buf-
fer to 1/3 the original volume. Proceed as described in
Subheading 3.4 .
3. The defrosting process of tissues high in levels of g-glutamyl
transpeptidase (e.g., kidneys, pancreas) will result in increased
g-glutamyl transpeptidase activity and poor experimental
results.
4. The UV-Visible plate reader can be set in the range of
405-414 nm.
5. Always do a dry run with the plate reader to ensure that the
GSH/GSSG ratio assay measurements are set up correctly.
This is especially important using the kinetic measurement.
Adding reagents and starting the reading only to find an
error in the set up is wasteful of time, tissue, and resources.
Checking the software will ensure time and materials are not
wasted.
6. Use the kinetic assay if not much is known about the contents
of the sample. The kinetic approach gives the most accurate
results if the sample contains many free thiols.
7. The endpoint and kinetic method calculations are multiplied
by two, in addition to sample dilutions, to account for pro-
tein deproteination steps.