Analysis of glutathione and glutathione disulfide in whole cells and mitochondria by postcolumn derivatization high-performance liquid chromatographywith ortho-phthalaldehyde

A method is described for the detection of glutathione (GSH) and glutathion e disulfide (GSSG) based on a HPLC postcolumn reaction with ortho-phthalald ehyde (OPT) at pH 12 followed by fluorescence detection. Although similar m ethods have been reported, the high pH of the postcolumn reaction adds cons iderable selectivity and sensitivity to the measurement of GSH and glutathi one disulfide. The limit of detection approaches 100 fmol, which is suffici ent to detect whole-cell glutathione disulfide in 10,000 cells or mitochond rial glutathione disulfide in 20 million cells. Using this method, glutathi one and glutathione disulfide were measured in human lymphocytes, granulocy tes, and cultured Jurkat T cells, as well as in the corresponding samples o f mitochondria, The percentage of glutathione disulfide to total glutathion e in whole-cell extracts was approximately 1%. In contrast, the percentage was relatively high in mitochondria, with the mitochondria of granulocytes having the highest (25%) followed by those of lymphocytes (15%) and finally by cultured Jurkat T cells (9%). This method extends the analysis of gluta thione and glutathione disulfide to mitochondria obtained from a relatively small number of cells. (C) 1999 Academic Press.