1 1 3 3 tetramethoxypropane

The malondialdehyde (MDA) content is an indicator of lipid peroxidation in the vital organs of animal samples. The level of lipid peroxidation from untreated and treated groups was assayed by thiobarbituric acid (TBA) reactive substance by the method of Garcia, Rodriguez-Malaver & Penaloza [32 (link)]. Briefly, the liver and kidney were harvested, meshed and filtered with the 70 μm cell strainers in PBS before centrifuged at 2000 rpm for 5 min. Then, 200 μL of supernatant was mixed with 800 μL of PBS, 25 μL of the 8.8% of butylated hydroxytoluene (BHT) and 500 μL of 30% of trichloroacetic acid (TCA), followed by incubation on ice for 2 h. After that, the mixtures were centrifuged at 2000 rpm and mixed with 250 μl of 1% thiobarbituric acid (TCA) and 75 μL of EDTA before they were heated to 95 °C for 15 min. Next, the mixture was chilled to room temperature and the absorbance was read at 532 nm using a spectrophotometer (Beckman Coulter, USA). The standard was prepared using 1,1,3,3-tetramethoxypropane (Sigma Aldrich, USA). The MDA concentration was calculated based on the standard curve of 1,1,3,3-tetramethoxypropane.

Acetylthiocholine iodide, 5,5’-dithiobis-2-nitrobenzoic acid (DTNB) were obtained from Merck (Germany), trichloroacetic acid (TCA), tris base, 1,1,3,3-tetramethoxypropane (MDA), 2-thiobarbituric acid (TBA), 2,4,6-tripyridyl-s-triazine (TPTZ), n-butanol, acetic acid, FeCl3-6H2O, benzethonium chloride (Hyamine® 1622), and phosphate buffer were acquired from Sigma-Aldrich (Germany). Analytical grade forms of CHP, DIA and MAL were obtained from local pesticide manufacturing companies. IMOD and ANG were supplied by Rose-Pharmed (Iran). The enzymelinked immunosorbent assay (ELISA) kits were purchased from Bender MedSystem (Germany).

Liver samples (0.25 g) were homogenized in 1 mL of 1.15% potassium chloride at 4 °C. The homogenates were centrifuged at 10,000× g for 10 min at 4 °C and the supernatants were used for analysis. Plasma and liver supernatants were previously deproteinized according to Pilz et al. [58 (link)]. LPO diene end-products, including malondialdehyde (MDA), were measured using a thiobarbituric acid (TBA) reaction [59 ]. Thiobarbituric acid reactive substances levels were measured spectrophotometrically at 535 nm (liver) or fluorometrically with excitation and emission wavelengths of 510 and 553 nm, respectively (plasma); 1,1,3,3-tetramethoxypropane (Sigma Aldrich, St. Louis, MO, USA) was used as standard. The results were expressed as µmol/L/g tissue (liver) or µmol/L of the plasma thiobarbituric acid reactive substances.

Pure fish oil was obtained from Zhejiang Shenzhou Marine Bioengineering Co., Ltd. (Zhoushan, Zhejiang, China). The composition of fatty acids (w/w) was determined by GC-MS and the total PUFAs content was 31.16%. WPI (protein content 92.9%) was commercial product of Hilmar Ingredients (Hilmar, CA, USA). Epicatechin (EC), 2,4-dinitrophenylhydrazine (DNPH), trichloroacetic acid (TCA), thiobarbituric acid (TBA), phosphate-buffered saline (PBS), phosphate-buffered saline (PBS), brilliant blue R250, 1,1,3,3-tetramethoxypropane, guanidine hydrochloride, and 5,5′-dithiobis (2-nitrobenzoic acid) were the products of Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan) or Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). 1,1,3,3-tetramethoxypropane, porcine pepsin (250 units/mg solid), and trypsin (8 USP size) were the products of Sigma-Aldrich Co. LLC (St. Louis, MO, USA). The rabbit polyclonal antibody to malondialdehyde-modified proteins (ab27642) and its corresponding secondary antibody was provided by Abcam (Cambridge, UK).

The following substances were used: sodium acetate, Alcian Blue, atropine, thiobarbituric acid, trichloroacetic acid, 5,5′-dithiobis (2-nitrobenzoic acid), bethanechol, carbenoxolone, 2,2-diphenyl-1-picrylhydrazyl, EDTA, glutathione, histamine, N-acetylcysteine, N-ethylmaleimide, nitro_ω-L-arginine methyl ester, pantoprazole, pentagastrin, ranitidine, sodium lauryl sulfate, thymol, 1,1,3,3-tetramethoxypropane (Sigma, St. Louis, USA), tris (hydroxymethyl) aminomethane, acetic acid, hydrochloric acid, ethanol, n-butanol, magnesium chloride, sodium chloride, potassium chloride, glucose, sodium hydroxide, anhydrous sodium sulfate, polysorbate 80 - Tween 80 (Vetec, Duque de Caxias, Brazil), ethyl ether, formaldehyde, phenolphthalein (FMaia, Cotia, Brazil), xylazine, ketamine (Vetbrands, Paulinia, Brazil). For the purposes of the experiment, the essential oil of Hyptis martiusii was emulsified in a Tween 80 at 1% before administration to the animals.