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

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 degrees 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.

Contents of D-lactate and its related metabolites as well as enzyme activities in the liver, muscle and blood plasma of aging rats.

As it is generally known. L-lactate is formed via the Embden-Meyerhof glycolytic pathway from triosephosphates, whereas D-lactate is formed via methylglyoxal in rat. In this paper, age-related changes in the levels of D-lactate and its related compounds in rat tissues are reported. Rats from 5 weeks to 30 months old were used in these experiments. (1) We observed that rats above 27 months old were decrepit as judged by external appearance movement and other physiological data of them. (2) The hepatic levels of D-lactate, methylglyoxal and pyruvate became markedly lower in aging rats, especially the D-lactate content in 30 month-old rats was lower by 90% than that of the 5 week-old rats. (3) As for plasma, D-lactate and phosphate levels became lower with aging, whereas levels of L-lactate and pyruvate were not altered. (4) In skeletal muscle, aging caused a lower methylglyoxal concentration. The D-lactate level was markedly decreased at the age of 30 months in muscle. (5) As for enzyme, activities of glyoxalase I and II became markedly decreased with age in livers, whereas the activity of glyoxalase I in muscle was maintained at control level and glyoxalase II increased with age.