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).