Assessment of antioxidant capacity and oxidative stress

The placental tissue was homogenised in 0.1 M phosphate buffer and centrifuged. Supernatants were collected and analysed for antioxidants and oxidative stress markers. Protein concentrations were determined using the Bio-Rad bicinchoninic acid (BCA) assay kit (Bio-Rad, Hercules, CA, USA).

Antioxidant capacity: The antioxidant capacity of the placental tissue was measured using the Trolox equivalent antioxidant capacity (TEAC) assay kit (Cayman Chemical Company, Ann Arbor, MI, USA), in accordance with the manufacturer's instructions.

Measurement of thiobarbituric acid reactive substances (TBARS): TBARS are oxidative stress markers that bind to malondialdehyde. TBARS levels in placental tissue were determined using the TBARS assay kit (Cayman Chemical Company) in accordance with the manufacturer's instructions.

Protein carbonyl measurement: The levels of oxidised proteins in tissue can be used as an indicator of oxidative stress. Protein carbonyl levels in the placental lysates were measured using the protein carbonyl colorimetric assay kit (Cayman Chemical Company). Protein carbonyls react with 2,4-dinitrophenylhydrazine to form a Schiff base that produces hydrazone, which is measured using a spectrophotometer set to quantify absorption at 385 nm.

Catalase activity: The activity of the antioxidant enzyme catalase was measured in placental tissue using a commercial assay kit (Cayman Chemical Company) in accordance with the manufacturer's instructions. The data were recorded as U/mg of tissue protein.