Enzymic method for quantitative determination of nanogram amounts of total and oxidized glutathione: Applications to mammalian blood and other tissues

A method for the analysis of nanogram quantities of glutathione has been developed which is based on the catalytic action of GSH or GSSG in the reduction of Ellman reagent (DTNB) by a mixture of TPNH and yeast glutathione reductase. Unlike previous methods of analysis the procedure described here effectively measures the total glutathione (GSH + GSSG) content of unknown mixtures and is not subject to appreciable interference by the presence of other thiol components. It is suggested that the catalytic action of glutathione in this system resides in the continual enzymic regeneration of GSH, present initially or formed enzymically from GSSG, following its interaction with the sulfhydryl reagent. The sensitivity of the method is such as to permit the determination of total glutathione in extracellular tissue fluids such as plasma, saliva, and urine normally containing very low levels of this material, essentially without pretreatment of the sample. The same is true for glutathione determinations of whole blood, in which the preliminary procedure is confined to the preparation of a 1:100 hemolyzate from as little as 10 μl of sample. Following published procedures, the pretreatment of tissue extracts with NEM to form an enzymically inactive complex with free GSH allowed the determination of the low levels of oxidized glutathione normally present therein. The use of the foregoing analytical method in the determination of total and oxidized glutathione contents of rat blood, kidney, and liver gave values in good agreement with those obtained by previous investigators.