Tetraethoxypropane

Trichloroacetic acid (TCA), thiobarbituric acid (TBA), phosphotungstic acid, tetraethoxypropane, nicotinamide adenine dinucleotide (NADH) and phenazine methosulfate (PMS), superoxide dismutase (SOD) standards, ethylenediaminetetraacetic acid (EDTA), nitro blue tetrazolium (NBT), phosphate-buffered saline (PBS), and 1% Triton X-100 were purchased from Sigma–Aldrich (St. Louis, MO, USA). Hydrochloric acid (HCl), acetic acid, sulfuric acid (H₂SO₄), magnesium chloride (MgC1₂), sodium hydroxide (NaOH), n-butanol, and hydrogen peroxide (H₂O₂) were purchased from Fisher Scientific (Chicago, IL, USA). The iron standard solution for AAS was purchased from Fluka (Buchs, Switzerland).

Lipid peroxidation was evaluated by measuring the amount of malondialdehyde (MDA) by the modified methods of Ohkawa based on TBA reactivity [33 (link)]. The standard curve is prepared with 1,1,3,3-tetraethoxypropane (Sigma–Aldrich, Munich, Germany) solution. Briefly, serum or hippocampal lysate (0.1 ml), SDS, and butylated hydroxytoluene were mixed and then TBA in acetate buffer (pH 3.0) was added. The tubes were incubated at 95 °C in a water bath for 60 min and after centrifugation, the supernatant was taken. After this, the solution’s adsorption was read at 532 and 572 nm, and the adsorption difference (A₅₃₂ – A₅₇₂) was used in the calculations.

In this study, we used the third-order derivative spectrophotometry method to determine the malondialdehyde (MDA) concentration in meat samples as described by Botsoglou, Fletouris [24 ]. At the end of the experiment, 4 birds in each pen (20 birds per treatment) were euthanized and deboned breast meat samples were collected. One gram of grounded meat sample was picked up and homogenized (Polytron homogenizer, PCU, Switzerland) with 4 ml of 5% aqueous trichloroacetic acid (TCA) and 2.5 ml of 0.8% butylated hydroxytoluene, and then centrifuged at 3000 × g for 3 min. The top layer (hexane) was discarded, and the bottom layer was filtered and made to 5 mL volume with 5% TCA, then placed into a screw-capped tube containing 3 mL of 0.8% aqueous 2-thiobarbituric acid (TBA). The tubes were heated at 70°C in a water bath for 30 min. After that, the tubes were cooled with tap water and then submitted to a spectrophotometer (UNIKON 933, Kontron Co. Ltd., Milan, Italy). The height of the third-order derivative peak that appeared at 521.5 nm was used to calculate the MDA concentration (expressed as milligrams per kilogram) as the secondary product of oxidation in the samples. The precursor of MDA in the standard curve was the tetraethoxypropane (1, 1, 3, 3- tetraethoxy propane, T9889, 97%, Sigma, USA).