Cs0410

The scavenging activity of 2,2-diphenyl-1-picrylhydrazine (DPPH) was analyzed using spectrophotometric methods [25 (link),26 (link)] with minor modifications. This study utilized the stable free radical DPPH (Sigma-Aldrich, St. Louis, MI, USA, Pcode: 101845869). In a 1.5-mL tube, 50 μL of each plasma and milk sample was mixed with 1 mL of DPPH reagent methanol solution (25 μmol/L). The mixture was thoroughly shaken and incubated in the dark at room temperature for 30 min, followed by centrifugation at 4000 r/min at 4 °C for 10 min. We then transferred the supernatant to a 96-well plate with 200 μL per well, and measured the absorbance at 517 nm using a microplate reader (Epoch, BioTek, Luzern, Switzerland). The DPPH-scavenging activity was calculated as a percentage using the following formula:
where Ac represents the control absorbance, and As represents the sample absorbance.
The enzyme activity of the total antioxidant capability (TAC), superoxide dismutase (SOD), glutathione S-transferase (GST), and malondialdehyde (MDA) was measured using Sigma-Aldrich kits, namely, MAK187 for TAC, 19160 for the SOD determination, CS0410 for GST, and MAK085 for MDA. Subsequently, the enzyme activity of all samples was determined using a microplate reader (Thermo Scientific™, Waltham, MA, USA).

During the NMP experiments, blood was collected from the NMP circuit at regular times for glutathione S-transferase (GST) activity analysis (see Figure 1a). After centrifugation for 15 min at 4 °C and 10,000× g, the supernatant was obtained and stored at −30 °C. Quantitative analysis of GST activity in blood plasma was performed using a GST assay kit (CS0410, Sigma Aldrich GmbH, Steinheim, Germany).

Glutathione S-transferase (GST, EC 2.5.1.18) activity was measured using a commercial kit (CS0410, Sigma). The conjugation of GSH to 1-chloro-2,4-dinitrobenzene (CDNB) catalyzed by GST was monitored at 340 nm for 4 min (SyneryTM HTX Multi-Mode). The reaction mixture contained 4 μL of extract and 196 μL of reaction solution (200 mM GSH and 100 mM CDNB in Dulbecco’s buffer at pH 7). The activity was calculated with ε = 9.6 mM−1 cm−1 [25 (link)]. A GST unit is defined as the amount of enzyme that catalyzes the formation of 1 μmol of the GS-DNB conjugate per minute at 25°C and pH 7. All reagents for oxidative stress detection were analytical grade from Sigma-Aldrich (St. Louis, MO, USA). The analysis were carried out on five subsamples for each site.