Assay Principle of Thiol/Disulfide Homeostasis Parameters
Total thiol and native thiol measurements were performed using Modified Ellman method of Erel et al. [10]. The reagent to be used for total thiol measurement was named 1 (R1) and the reagent to be used for native thiol measurement (R1’). While these initial reagents were different in total thiol and native thiol measurements, the other reagents were the same. R1 was used freshly prepared on the day of the study, with a final concentration of 378 mg of sodium borohydrate (NaBH4) in 1000 mL of water-methanol solution (with a volume ratio of 1/1) of 10 mM. This reducing solution was used to determine the total thiol content. R1’ 585 mg of sodium chloride (NaCl) in 1000 mL of water-methanol solution (1/1 volume ratio) was prepared freshly on the day of the study, with a final concentration of 10 mM. The obtained reagent was stored at +4 °C for 6 months. This solution was used to determine the native thiol amount. Reagent 2 (R2) was prepared freshly by dissolving 0.5 mL of formaldehyde with a final concentration of 6.715 mM and 3.8 g of EDTA with a final concentration of 10 mM in 1000 mL of Tris buffer, 100 mM and pH 8.2. The obtained reagent was stored at +4 °C for 6 months. This solution was used for total and native thiol measurements. As to Reagent 3 (R3) 3.963 g of 5,5-dithiobis-2-nitrobenzoic acid (DTNB) was prepared freshly on the working day at a final concentration of 10 mM in 1000 mL methanol. This solution was used for total and native thiol measurements.
Total and native thiol measurement principle: For the total thiol amount measurement, 10 μl R1 (10 μl R1 native for native thiol amount measurement) and 10 μl sample were mixed. Then, the first absorbance reading (A1) was performed spectrophotometrically at 415 nm wavelength by adding R2 and R3 (Schimadzu UV-1800 spectrophotometer, Kyoto, Japan). The second absorbance (A2) reading was made at the 10th minute of the reaction at the same wavelength. Absorbance difference (A2-A1) was obtained and the measurement was completed. To determine the total thiol and native thiol levels, the molar extinction coefficient of 5-thio-2-nitrobenzoic acid (TNB) was 14.100 mol / L-1 cm-1. Measurement of disulfide level was calculated using the formula [(total thiol-native thiol) / 2].
We also calculated corrected native thiol, total thiol and disulfide levels based on the serum albumin concentrations from the following formulas:
Corrected total thiol levels: total thiol (µmol/L)/ albumin (g/L).
Corrected native thiol levels: native thiol (µmol/L)/ albumin (g/L).
Corrected disulfide levels: disulfide (µmol/L) /albumin (g/L).