Nicotinamide adenine dinucleotide phosphate-regenerating system coupled to a glutathione-reductase microtiter method for determination of total glutathione concentrations in adherent growing cancer cell lines

Abstract

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 μM), 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 μM d, l-buthionine- S, R-sulfoximine (BSO), an inhibitor of GSH biosynthesis. As expected, BSO lowered GSH levels on the average 85%.