Improvements in the traditional
glutathione (GSH)-
reductase recycling method for determining total
glutathione levels in adherent growing cells have been achieved by eliminating the direct use of expensive
nicotinamide adenine dinucleotide phosphate (
NADPH) and normalizing the levels of GSH to moles/liter instead of the more usual but more error-prone method of normalizing with cellular
protein. A glucose-6-phosphate-dehydrogenase auxiliary reaction has been added to the microtiter-adapted
enzyme method of Tietze; thus
NADP(+) and
glucose-6-phosphate replace
NADPH in the method. This modification lowers the possibility for substrate inhibition of the
reductase by high levels of
NADPH during the initial phase of the reaction while at the same time reducing the assay costs by 75-85%. To calculate the cellular concentration of GSH, the number of cells used for the GSH determination, estimated by counting cell nuclei of
benzalkonium chloride-lysed cells with a Coulter Counter Z2, and the average cell volume, also determined with the Coulter Counter, are multiplied to give the total sample volume. The quotient of the amount of GSH found in the cells and the total sample volume yields the GSH concentration in moles/liter. The assay has been validated with respect to precision (+/-2.6%), relative accuracy (-4.2 %), linearity (r(2)=0.98), linear range (0.5-10 microM), and limit of detection (80 pmol). Recovery was cell line dependent and ranged between 70 and 103% in the six cell lines. As an application of this method, the GSH concentrations in six human
cancer cell lines were determined, without and with a 24-h preincubation with 200 microM D,L-
buthionine-S,R-sulfoximine (BSO), an inhibitor of GSH biosynthesis. As expected, BSO lowered GSH levels on the average 85%.