Ultra turrax

Key brain regions such as the prefrontal cortex, hippocampus, and striatum were carefully dissected and then homogenized in an extraction solution (100 mg of tissue per milliliter), containing 0.4 M NaCl, 0.05% Tween 20, 0.5% BSA, 0.1 mM phenylmethylsulphonyl fluoride, 0.1 mM benzethonium chloride, 10 mM EDTA, and 20 KIU aprotinin, using Ultra-Turrax (IKA®-Werke GmbH & CO, Staufen, Alemanha). Lysates were centrifuged at 13,000× g for 10 min at 4 °C and supernatants were collected. Samples were then thawed and the tissue levels of ACE, Ang II, ACE2, and Ang-(1-7) were measured by ELISA, according to the procedures supplied by the manufacturer (MyBioSource, San Diego, CA, USA). All kits applied the sandwich ELISA technique, except for the ACE measurement, whose kit applied the competitive ELISA method. The sensitivity of the assay was 10 pg/mL for ACE and ACE2; 12 pg/mL for Ang II; and 1.5 pg/mL for Ang-(1-7).

P_OX, measured as total carbonyl content, were quantified according to the method described by Oliver [19 (link)] and modified by Vuorela [20 (link)], commonly known as the DNPH method. Each of the meat samples was homogenized with 20 mL of 0.6 M NaCl for 60 s using an Ultra-Turrax homogenizer (IKA T25 digital Ultra-Turrax, IKA-Werke GmbH & Co., Staufen im Breisgau, Germany). Two aliquots of homogenate were taken (0.1 mL) and were transferred into Eppendorf vials. Proteins were precipitated with 10% (1 mL) TCA and were centrifuged for 5 min at 10,000× g. One of the pellets was treated with 2N HCl (1 mL) in order to quantify proteins and the other one with 0.2% 2,4-dinitrophenyl hydrazine (DNPH) in HCl 2M (1 mL) to quantify carbonyls. Part of the extract was used for MIR measurement (P_OX marker compound). Protein concentration samples (p) were measured spectrophotometrically attending to absorbance at 280 nm (Spectrophotometer UV/vis with diode detector, Shimadzu UV-2101PC, Mettler-Toledo S.A.E. L’Hospitalet de Llobregat, Barcelona, Spain) using Bovine Serum Albumin (BSA) as the standard. Carbonyl content was expressed as nmol of carbonyl compounds per milligram of protein using an extinction coefficient of 21.0 mM⁻¹ cm⁻¹ at 370 nm.

Malondialdehyde content was measured following the procedure of Hodges et al. (1999): 0.2 g of frozen plant material was homogenized in 2 ml of 80% cold ethanol (Panreac) using a tissue homogenizer (Ultra‐Turrax; IKA‐Werke). Homogenates were centrifuged at 4,500 rpm for 20 min at 4°C to pellet debris, and different aliquots of the supernatant were mixed either with 20% trichloroacetic acid (Panreac) or with a mixture of 20% trichloroacetic acid and 0.5% thiobarbituric acid (Sigma‐Aldrich). Both mixtures were incubated in a water bath at 90°C for 1 hr.
Afterward, the samples were centrifuged. The absorbance of the supernatant was read at 440, 534, and 600 nm against a blank. The MDA concentration in the extracts was calculated according to Arbona and Gomez‐Cadenas (2008).