2.5. Lipid Peroxidation Assay

The colorimetric lipid peroxidation assay kit (ab118970, Abcam, Cambridge, United Kingdom) is a suitable tool for the detection of MDA in a variety of samples. The MDA present in the sample reacts with thiobarbituric acid (TBA) to generate MDA-TBA, which is colorimetrically quantified (OD 532 nm). This assay detects MDA levels from 0.1 mol/well MDA (1 mol/well MDA in colorimetry). This study quantified the levels of MDA in umbilical cord tissue samples and the blood plasma of newborns. To perform the experiment, the following protocol was used: (1–3) MDA lysis buffer, phosphotungstic acid and butylated hydroxytoluene (BHT) solution (100×), ready-to-use; the buffer was stored at −20 °C and brought to room temperature before use; (4) TBA solution: A vial of TBA was reconstituted in 7.5 mL of glacial acetic acid and then transferred to another tube, where the final volume was adjusted to 25 mL with distilled water; the sample was mixed well to dissolve the TBA, sonication was performed in an RT water bath and the sample was stored at 4 °C; and (5) MDA standard (4.17 M), ready-to-use: The solution was stored at −20 °C and brought to room temperature before use.

Tissue samples: A total of 20 mg of umbilical cord tissue was used for the analysis. The protocol used was as follows: (1) The tissue was rinsed in cold PBS; (2) the lysis solution (300 mL of lysis buffer with MDA and 3 µL of BHT (100×)) was prepared; (3) the tissue was homogenized in 303 µL of lysis solution (buffer + BHT) using 10–15 passes with a homogenizer on ice; and (4) centrifugation was performed at 13,000× g for 10 min to remove the insoluble material and collect the supernatant.

Plasma samples: Samples of 10 µL of blood plasma were used for the two study periods (32 weeks of gestation and 32 weeks after delivery). The protocol used was as follows: (1) Carefully mix 10 µL of plasma with 500 µL of 42 mM H₂SO₄ in a microcentrifuge tube; (2) add 125 µL of phosphotungstic acid solution and mix by vortexing; (3) incubate at room temperature for 5 min; (4) centrifuge at 13,000× g for 3 min; (5) collect the precipitate and resuspend on ice with 100 µL of distilled water (with 2 µL of BHT (100×)); and (6) adjust the final volume to 200 µL with distilled water.

For the colorimetric assay, a 0.1-M MDA standard was prepared by diluting 10 µL of 4.17 M standard MDA in 407 µL of distilled water. A 2-mM MDA standard was prepared by diluting 10 µL of 0.1 M standard MDA in 490 µL of distilled water. The MDA standard (2 mM) was used, and dilutions were prepared for the standard curve. MDA-TBA was generated by adding 600 µL of TBA to each vial. The samples were incubated at 95 °C for 60 min. Each sample was cooled to room temperature in an ice bath for 10 min. Then, 200 µL of the TBA/standard mixture and 200 µL of the TBA/sample mixture were combined in a 96-well plate for analysis. For greater sensitivity, 300 µL of n-butanol was added to the MDA-TBA precipitate. If there was no separation, 100 µL of 5 M NaCl was added to the mixture and then vigorously stirred. The layers could then be separated by centrifugation (3 min at 16,000× g). The MDA-TBA phase was transferred to a new tube, and the n-butanol was evaporated. MDA-TBA was dissolved in 200 µL of distilled water and then placed in a 96-well plate for analysis. The product was immediately measured in a reader at OD 532 nm for the colorimetric assay. The measurement was performed using a Victor 2 multifunction device (Wallac, Victoria, Australia).