ABSTRACT
The peroxyoxalate chemiluminescence detection of biological thiols combined with high-performance liquid chromatography (HPLC) is described. SH groups of the thiol compounds including glutathione (GSH), cysteine, N-acetylcysteine, cysteamine, and D-penicillamine were labelled with N-[4-(6-dimethylamino-2-benzofuranyl)phenyl]maleimide (DBPM), a specific fluorogenic reagent for SH group. The labelling reaction was carried out at 60 degrees C for 30 min and at pH 8.5 and a sample of the resulting reaction mixture was subjected to HPLC. Five kinds of labelled thiols were separated within 12 min on ODS-80 column (150 x 4.6 mm ID; 5 microns) and detected in the ranges from 500 fmol to 2 pmol/100 microL (cysteamine and N-acetylcysteine), to 3 pmol/100 microL (cysteine) and to 5 pmol/100 microL (GSH and D-penicillamine). The lower detection limits were from 7 fmol (cysteamine) to 113 fmol (GSH) per 100 microL (S/N = 2). The method was applied to the determination of thiols in a rat liver. The amounts of glutathione and cysteine were 1.23 +/- 0.15 mumol/g (n = 5) and 0.15 +/- 0.04 mumol/g (n = 5), respectively.
Subject(s)
Sulfhydryl Compounds/analysis , Animals , Benzofurans , Chromatography, High Pressure Liquid , Fluorescent Dyes , Liver/analysis , Luminescent Measurements , Male , Maleimides , Rats , Rats, Inbred StrainsABSTRACT
The selective determination of thiols in biological samples was investigated by high-performance liquid chromatography using N-[4-(6-dimethylamino-2-benzofuranyl)phenyl] maleimide, which was found to give fluorescent products when treated with certain thiols. Six kinds of thiol (reduced glutathione, cysteine, N-acetylcysteine, cysteamine, homocysteine and coenzyme A) could be separated simultaneously within ca. 12 min and determined at final level of sensitivity. The method was successfully applied to the determination of thiols in rat tissues and plasma and in human normal serum.
