N methyl 2phenyl indole

Malondialdehyde (MDA) and 4-hydroxyalkenal (4-HDA) concentration was measured (n = 5) as described previously [32 (link)]. The colorimetric reaction was triggered in 200 μL of the supernatant after the addition of 650 μL of 10.3 mM N-methyl-2phenyl-indole (Sigma-Aldrich; Saint Louis, MO, USA) diluted in a mixture of acetonitrile : methanol (3 : 1) and 150 μL of methanesulfonic acid (Sigma-Aldrich; Saint Louis, MO, USA). The reaction mixture was vortexed and incubated at 45°C for 1 h and afterward centrifuged at 3000 rpm for 10 min. The absorbance was read at 586 nm in the supernatant with a SmartSpec 3000 spectrophotometer (Bio-Rad; Hercules, CA, USA). The absorbance values were compared to a standard curve of 0.25 to 5 μM of 1,1,3,3-tetramethoxypropane (10 mM stock) to calculate the content of MDA and 4-HDA in the samples. The values were expressed as the nM MDA and 4-HDA/mg of protein.

Malondialdehyde (MDA) and 4-hydroxyalkenals (4-HAD) were measured in the same supernatant where the nitrites were measured (n = 5 rats in each group), following the procedure described elsewhere [22 (link)]. The colorimetric reaction was made using 200 μL of the supernatant after the subsequent addition of 650 μL of 10.3 mM N-methyl-2phenyl-indole (Sigma-Aldrich; Saint Louis, MO, USA) diluted in a mixture of acetonitrile : methanol (3 : 1) and 150 μL of methanesulfonic acid (Sigma-Aldrich; Saint Louis, MO, USA). The reaction mixture was vortexed and incubated at 45°C for 1 h and afterward centrifuged at 3000 rpm for 10 min. The absorbance in the supernatant was read at 586 nm with a SmartSpec 3000 spectrophotometer (Bio-Rad; Hercules, CA, USA). The absorbance values were compared to a standard curve in the concentration range of 0.5 to 5 μM of 1,1,3,3-tetramethoxypropane (10 mM stock) to calculate the content of malondialdehyde + 4-hydroxyalkenal (MDA + 4-HAD) in the samples.

The nitric oxide reacts with the superoxide anion to produce peroxynitrite, which causes the oxidation of lipids and can be detected by the Gérard-Monnier method. Malondialdehyde (MDA) and 4-hydroxyalkenals (4-HDA) were measured at 34 days post-reperfusion in the same supernatant used for nitrite detection of (n = 5 rats per group) as described elsewhere [62 (link)]. To generate the colorimetric reaction, 200 μL of the supernatant was mixed with 650 μL of 10.3 mM N-methyl-2phenyl-indole (Sigma-Aldrich; Saint Louis, MO, USA), diluted in a mixture of acetonitrile and methanol in a 3:1 ratio. Further, 150 μL of methanesulfonic acid (Sigma-Aldrich; Saint Louis, MO, USA) was added to the mixture. After the reaction mixture was vortexed, it was incubated at 45 °C for 1 h. Following this, it was centrifuged at 3000 rpm for 10 min. The sample was then measured at 586 nm using a Smart-Spec 3000 spectrophotometer (Bio-Rad; Hercules, CA, USA). A standard curve was compared in the concentration range of 0.5–5 μM of 1,1,3,3-tetramethoxypropane (10 mM stock) to determine the content of MDA + 4-HDA in each sample.