Multiparametric protocol for the determination of thiol redox state in living matter

• Assessment of TRS is difficult because of its multiparametric nature.
• Existing methods assess TRS partially and nonspecifically.
• The present protocol covers the need for a complete TRS evaluation.
• It simultaneously quantifies 9 nonprotein and 6 protein TRS parameters.

Thiol redox state (TRS) evaluation is mostly restricted to the estimation of GSH and GSSG. However, these TRS parameters can estimate the GSSG/GSH potential, which might be useful for indicating abnormalities in redox metabolism. Nonetheless, evaluation of the multiparameric nature of TRS is required for a more accurate assessment of its physiological role. The present protocol extends the partial assessment of TRS by current methodologies. It measures 15 key parameters of TRS by two modular subprotocols: one for the glutathione (GSH)- and cysteine (CSH)-based nonprotein (NP) thiols/mixed disulfides (i.e., GSH, GSSG, GSSNP, CSH, CSSNP, NPSH, NPSSNP, NPxSHNPSSNP, NPxSHNPSH), and the other for their protein (P) thiols/mixed disulfides (i.e., PSH, PSSG, PSSC, PSSNP, PSSP, NPxSHPSSNP). The protocol eliminates autoxidation of GSH and CSH (and thus overestimation of GSSG and CSSNP). Its modularity allows the determination GSH and GSSG also by other published specific assays. The protocol uses three assays; two are based on the photometric reagents 4,4′-dithiopyridine (DTP) and ninhydrin (NHD), and the third on the fluorometric reagent o-phthaldialdehyde (OPT). The initial assays employing these reagents have been extensively modified and redesigned for increased specificity, sensitivity, and simplicity. TRS parameter values and their standard errors are estimated automatically by sets of Excel-adapted algebraic equations. Protocol sensitivity for NPSH, PSH, NPSSNP, PSSP, PSSNP, CSH, CSSNP, PSSC, NPxSHNPSSNP, and NPxSHNPSH is 1 nmol –SH/CSH, for GSSNP 0.2 nmol, for GSH and GSSG 0.4 nmol, and for PSSG 0.6 nmol. The protocol was applied on human plasma, a sample of high clinical value, and can be also applied in any organism.

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