The static ORP (sORP) marker was determined using the RedoxSYS diagnostic system (Luoxis Diagnostics, Inc., Englewood, CO, USA) as previously described (17 (link),18 (link)). This value is indicative of the integrated balance of oxidants and reductants in a specimen and is presented in mV. Using this innovative method, 20 µl of plasma were applied to disposable sensors designed by Luoxis Diagnostics, Inc., which were inserted into the RedoxSYS diagnostic system and the sORP value was reported within 4 min.
For the determination of the levels of TBARS, an assay was used based on the study by Keles et al (19 (link)). TBARS is a commonly and frequently used method to determine the lipid peroxidation (20 (link)). In accordance with this method, 100 µl of plasma were mixed with 500 µl of 35% trichloroacetic acid (Merck KGaA, Darmstadt, Germany) and 500 µl of Tris-HCl (Sigma-Aldrich, St. Louis, MO, USA; 200 mmol/l, pH 7.4) followed by incubation for 10 min at room temperature. A total of 1 ml of 2 M sodium sulfate and 55 mmol/l TBA solution were added and the samples were then incubated at 95°C for 45 min. The samples were cooled on ice for 5 min and were vortexed following the addition of 1 ml of 70% TCA. The samples were centrifuged at 15,000 × g for 3 min and the absorbance of the supernatant was read at 530 nm using a spectrophotometer (Hitachi U-1900; serial no. 2023-029; Hitachi, Tokyo, Japan). A baseline absorbance was taken into account by running a blank along with all samples during the measurement. The calculation of the TBARS concentration was based on the molar extinction co-efficient of malondialdehyde.
The GSH concentration was measured as previously described in the study by Reddy et al (21 ). A total of 20 µl of erythrocyte lysate treated with 5% TCA was mixed with 660 µl of 67 mmol/l sodium potassium phosphate (pH 8.0) and 330 µl of 1 mmol/l 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB). The samples were then incubated in the dark at room temperature for 45 min and the absorbance was read at 412 nm using a spectrophotometer (Hitachi U-1900; serial no. 2023-029; Hitachi). The GSH concentration was calculated on the basis of calibration curves made using commercial standards.
The concentration of CARB, an index of protein oxidation, was determined based on the method described in the study by Patsoukis et al (22 (link)). In this assay, 50 µl of 20% TCA were added to 50 µl of plasma and this mixture was then incubated in an ice bath for 15 min and centrifuged at 15,000 × g for 5 min at 4°C. The supernatant was discarded and 500 µl of 10 mmol/l 2,4-dinitrophenylhydrazine (DNPH; in 2.5 N HCl) for the sample, or 500 µl of 2.5 N HCl for the blank, were added to the pellet. The samples were incubated in the dark at room temperature for 1 h with intermittent vortexing every 15 min and were centrifuged at 15,000 × g for 5 min at 4°C. The supernatant was discarded and 1 ml of 10% TCA was added, vortexed and centrifuged at 15,000 × g for 5 min at 4°C. The supernatant was discarded and 1 ml of ethanol-ethyl acetate (1:1 v/v) was added, vortexed and centrifuged at 15,000 × g for 5 min at 4°C. This washing step was repeated twice. The supernatant was discarded and 1 ml of 5 M urea (pH 2.3) was added, vortexed and incubated at 37°C for 15 min. The samples were centrifuged at 15,000 × g for 3 min at 4°C and the absorbance was read at 375 nm using a spectrophotometer (Hitachi U-1900; serial no. 2023-029; Hitachi). The calculation of the CARB concentration was based on the molar extinction co-efficient of DNPH. Total plasma protein was assayed using Bradford reagent (Sigma, Hamburg, Germany).
The determination of TAC was based on the method described in the study by Janaszewska and Bartosz (23 (link)). Briefly, 20 µl of plasma were added respectively to 480 µl of 10 mmol/l sodium potassium phosphate (pH 7.4) and 500 µl of 0.1 mmol/l 1,1-diphenyl-1-picrylhydrazyl (DPPH) and the samples were incubated in the dark for 60 min at room temperature. The samples were then centrifuged for 3 min at 20,000 × g and the absorbance was read at 520 nm using a spectrophotometer (Hitachi U-1900; serial no. 2023-029; Hitachi).
The measurement of CAT activity was carried out as previously described by Aebi (24 (link)). In particular, 4 µl οf erythrocyte lysate (diluted 1:10) were added to 2,991 µl οf 67 mmol/l sodium potassium phosphate (pH 7.4) and the samples were incubated at 37°C for 10 min. A total of 5 µl of 30% H2O2 was added to the samples and the change in absorbance was immediately read at 240 nm [using a spectrophotometer (Hitachi U-1900; serial no. 2023-029; Hitachi)] for 130 sec. The calculation of CAT activity was based on the molar extinction co-efficient of H2O2. Each assay was performed twice in triplicate.
For the determination of the levels of TBARS, an assay was used based on the study by Keles et al (19 (link)). TBARS is a commonly and frequently used method to determine the lipid peroxidation (20 (link)). In accordance with this method, 100 µl of plasma were mixed with 500 µl of 35% trichloroacetic acid (Merck KGaA, Darmstadt, Germany) and 500 µl of Tris-HCl (Sigma-Aldrich, St. Louis, MO, USA; 200 mmol/l, pH 7.4) followed by incubation for 10 min at room temperature. A total of 1 ml of 2 M sodium sulfate and 55 mmol/l TBA solution were added and the samples were then incubated at 95°C for 45 min. The samples were cooled on ice for 5 min and were vortexed following the addition of 1 ml of 70% TCA. The samples were centrifuged at 15,000 × g for 3 min and the absorbance of the supernatant was read at 530 nm using a spectrophotometer (Hitachi U-1900; serial no. 2023-029; Hitachi, Tokyo, Japan). A baseline absorbance was taken into account by running a blank along with all samples during the measurement. The calculation of the TBARS concentration was based on the molar extinction co-efficient of malondialdehyde.
The GSH concentration was measured as previously described in the study by Reddy et al (21 ). A total of 20 µl of erythrocyte lysate treated with 5% TCA was mixed with 660 µl of 67 mmol/l sodium potassium phosphate (pH 8.0) and 330 µl of 1 mmol/l 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB). The samples were then incubated in the dark at room temperature for 45 min and the absorbance was read at 412 nm using a spectrophotometer (Hitachi U-1900; serial no. 2023-029; Hitachi). The GSH concentration was calculated on the basis of calibration curves made using commercial standards.
The concentration of CARB, an index of protein oxidation, was determined based on the method described in the study by Patsoukis et al (22 (link)). In this assay, 50 µl of 20% TCA were added to 50 µl of plasma and this mixture was then incubated in an ice bath for 15 min and centrifuged at 15,000 × g for 5 min at 4°C. The supernatant was discarded and 500 µl of 10 mmol/l 2,4-dinitrophenylhydrazine (DNPH; in 2.5 N HCl) for the sample, or 500 µl of 2.5 N HCl for the blank, were added to the pellet. The samples were incubated in the dark at room temperature for 1 h with intermittent vortexing every 15 min and were centrifuged at 15,000 × g for 5 min at 4°C. The supernatant was discarded and 1 ml of 10% TCA was added, vortexed and centrifuged at 15,000 × g for 5 min at 4°C. The supernatant was discarded and 1 ml of ethanol-ethyl acetate (1:1 v/v) was added, vortexed and centrifuged at 15,000 × g for 5 min at 4°C. This washing step was repeated twice. The supernatant was discarded and 1 ml of 5 M urea (pH 2.3) was added, vortexed and incubated at 37°C for 15 min. The samples were centrifuged at 15,000 × g for 3 min at 4°C and the absorbance was read at 375 nm using a spectrophotometer (Hitachi U-1900; serial no. 2023-029; Hitachi). The calculation of the CARB concentration was based on the molar extinction co-efficient of DNPH. Total plasma protein was assayed using Bradford reagent (Sigma, Hamburg, Germany).
The determination of TAC was based on the method described in the study by Janaszewska and Bartosz (23 (link)). Briefly, 20 µl of plasma were added respectively to 480 µl of 10 mmol/l sodium potassium phosphate (pH 7.4) and 500 µl of 0.1 mmol/l 1,1-diphenyl-1-picrylhydrazyl (DPPH) and the samples were incubated in the dark for 60 min at room temperature. The samples were then centrifuged for 3 min at 20,000 × g and the absorbance was read at 520 nm using a spectrophotometer (Hitachi U-1900; serial no. 2023-029; Hitachi).
The measurement of CAT activity was carried out as previously described by Aebi (24 (link)). In particular, 4 µl οf erythrocyte lysate (diluted 1:10) were added to 2,991 µl οf 67 mmol/l sodium potassium phosphate (pH 7.4) and the samples were incubated at 37°C for 10 min. A total of 5 µl of 30% H2O2 was added to the samples and the change in absorbance was immediately read at 240 nm [using a spectrophotometer (Hitachi U-1900; serial no. 2023-029; Hitachi)] for 130 sec. The calculation of CAT activity was based on the molar extinction co-efficient of H2O2. Each assay was performed twice in triplicate.
