A colorimetric determination of total glutathione based on its C-terminal glycine residue and its application to blood,liver and yeast.

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Abstract
An assay procedure for total glutathione in blood, liver and yeast was developed, based on the color reaction of the glycine residue, not the SH group. Glycine, which showed a color reaction similar to that of glutathione, was first eliminated by reaction with sodium nitrite under acidic conditions, then deaminated glutathione was reacted with benzoyl chloride in NaOH solution. The reaction product(s) was extracted with ethyl acetate containing 5% (vol/vol) ethanol, after acidification of the reaction mixture with phosphoric acid. An aliquot of the organic phase was evaporated to dryness under reduced pressure, and the dried residue was treated to develop the color by the addition of acetic anhydride, p-dimethylaminobenzaldehyde and pyridine. The absorbance was measured at 458 nm after reaction at 40.degree. for 1 h. Beer''s law was obeyed in the range from 0.05-1.0 .mu.mol of the reduced form or from 0.025-0.5 .mu.mol of the oxidized form in the curvette. This procedure is applicable to the determination of total glutathione in amino acid mixtures, whole blood, rat liver cytosol and yeast heat extract.