High-performance liquid chromatographic method to analyzed picomole levels of glutathione, cysteine nd cysteinylglycine and its application to pre-cancerous rat livers

摘要

A HPLC-based method for quantifying glutathione, cysteine and cysteine-containing peptide is described. N-(2,4-Dinitrophenylaminoethyl)maleimide was synthesized from ethylenediamine, 2,4-dinitrochlorobenzene and maleic anhydride. The maleimide was reacted at 40 deg C and pH 5.8 for 10 min with thiol compounds such as glutathione or cysteine. An aliquot of the reaction mixtures was applied to a reversed-phase column (4.6 X 150 mm) of HPLC. When glutathione and cysteine were simultaneously assayed, the column was eluted with a gradient of acetonitrile in potassium phosphate (pH 7.0). The derivatives were monitored at 350 nm. Good liner relationships existed between peak area and concentration of glutathione or cysteine from 10 pmol to 2 nmol. The recovery tests from rat liver homogenate were 99.7 +- 2.2% for glutathione and 104.9 +- 3.8% for cysteine. By this method #gamma#-glutamylcysteine, cysteinylglycine, and homocysteine could be also quantified. The determination limits of glutathione, cysteine and other thiol compounds were 5 pmol. The method is simple: a sample solution is mixed with the labelling reagent and an aliquot of the reaction mixture is applied to a standard HPLC. The hepatic levels of cysteine and glutathione in pre-cancerous rats were determined by this method. The cysteine level in pre-cancerous livers was extremely elevated in comparison to that of the control groups, while no difference was observed in the glutathione contents between the pre-cancerous and control groups.