A novel and automated assay for thiol/disulphide homeostasis

Abstract

ObjectivesTo develop a novel and automated assay determining plasma thiol/disulphide homeostasis, which consists of thiol–disulphide exchanges. Design and methodsNative thiol and total thiol concentrations were synchronously measured as a paired test. In the first vessel, the amount of native thiol groups was measured by a modified Ellman reagent. At the parallel run, first, dynamic disulphide bonds were reduced to free thiol groups by NaBH4. The unused reductant remnants were completely removed by formaldehyde. Thus, the total thiol amount could be accurately measured. Mercaptoethanol solutions were used as calibrators. The half value of the difference between total thiol and native thiol amounts gave the disulphide bond amount. ResultsNo separation step for the assay was needed. All processes were performed using an automated analyser within about 10min. Plasma disulphide levels were 17.29±5.32μmol/L, native thiol levels were 397±62μmol/L and disulphide/native thiol per cent ratios were 4.32±1.49 in healthy subjects. Plasma disulphide levels were higher in patients with degenerative diseases and lower in patients with proliferative diseases. ConclusionAn easy, inexpensive, practical, fully automated and also optionally manual spectrophotometric assay can be used to determine plasma dynamic thiol/disulphide homeostasis.