A novel and automated assay for thiol/disulphide homeostasis

• Published in: Clinical biochemistry Vol. 47; no. 18; pp. 326 - 332
• Main Authors: Erel, Ozcan, Neselioglu, Salim
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.12.2014
• Subjects: Borohydrides - chemistry; Chemistry Techniques, Analytical - methods; Disulfides - blood; Disulfides - chemistry; Disulphide; Formaldehyde - chemistry; Glutathione - chemistry; Homeostasis; Humans; Mercaptan; Mercaptoethanol - chemistry; Oxidation-Reduction; Reproducibility of Results; Serum Albumin - chemistry; Sulfhydryl; Sulfhydryl Compounds - blood; Sulfhydryl Compounds - chemistry; Thiol; GSSG; Thiol; NaBH4; DTNB; Mercaptan; RSSR,–S–S; Sulfhydryl; SH; CySS; Disulphide; Cys; 4-DPS; GSH; TCEP
• ISSN: 0009-9120; 1873-2933; 1873-2933
• DOI: 10.1016/j.clinbiochem.2014.09.026

To develop a novel and automated assay determining plasma thiol/disulphide homeostasis, which consists of thiol–disulphide exchanges. Native 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. No 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. An easy, inexpensive, practical, fully automated and also optionally manual spectrophotometric assay can be used to determine plasma dynamic thiol/disulphide homeostasis. •A novel assay determining plasma thiol/disulphide homeostasis.•The plasma thiol amount was measured.•The plasma dynamic disulphide bond amount was measured.•Plasma dynamic disulphide bond amounts increase in degenerative diseases.•Plasma dynamic disulphide bond amounts decrease in proliferative diseases.