ABC | Volume 113, Nº5, November 2019

Original Article Muniz et al. Metabolic disorders in a modified dyslipidemia model Arq Bras Cardiol. 2019; 113(5):896-902 seven weeks (one week of acclimation and six weeks of assay), under controlled environmental conditions (12 h light, 12 h dark cycle, and temperature at 22 ± 2ºC). In the experiment, we randomly divided 24 rats into 3 groups of 8 animals, according to a randomized block design based on their body weight. The sample size was defined considering previous studies on experimental dyslipidemia. Each group received one of the following diets: standard diet (STD; RH19521, Rhoster, Brazil) prepared according to AIN-93M, 13 high-lard diet (HLD; RH195143, Rhoster, Brazil), and high-lard and high‑cholesterol diet (HL/HCD; RH195142, Rhoster, Brazil). Soybean oil (STD) and lard were used as lipid sources; cholesterol, to induce hypercholesterolemia; and cholic acid, to increase the hypercholesterolemic effect of supplemented cholesterol 14 (Table 1). The diets and filtered water were provided ad libitum . Dietary intake and body weight of the animals were monitored three times a week. After six weeks of experimentation, the animals were euthanized (xylazine + ketamine/ 10 mg/kg + 100 mg/kg). Next, we collected blood samples by abdominal puncture (about 5 mL) for biochemical analyses (serum lipid profile and liver enzymes). After the euthanasia by abdominal aorta exsanguination, we extracted, weighed, and prepared the liver of the animals for morphological evaluation. All procedures complied with the Guide for the Care and Use of Laboratory Animals 15 and were approved by the Animal Care and Use Ethics Committee of the Federal University of Goiás (protocol number 039/15). Biochemical analyses Blood samples were collected in heparin tubes and centrifuged at 4000 rpm for 10 min to separate the serum, which was immediately stored at –80°C for analyses of lipid profile and liver enzymes. Total cholesterol (TC), TG, LDL-c, HDL-c, alanine aminotransferase (ALT), and aspartate aminotransferase (AST) levels were determined by commercially available kits (Labtest Diagnóstica S.A., Lagoa Santa, Brazil). Very low-density lipoprotein cholesterol (VLDL-c) was estimated by the Friedewald equation, and non‑HDL-c fraction was obtained by the difference between TC and HDL-c levels. The importance of non-HDL-c fraction has been highlighted in cardiovascular disease (CVD) risk prediction. Boekholdt et al. 16 revealed that non-HDL-c concentrations had a stronger direct association with CVD risk than LDL-c and apolipoprotein B (Apo B). Liver morphological evaluation We rinsed the liver lobes in ice-cold 0.9% physiological solution and fixed them by immersion in phosphate‑buffered 10% formalin for 24 h. Subsequently, the lobes were cut into 4 to 5 mm thick fragments, dehydrated in increasing alcohol concentrations (80%, 95%, and 100%), clarified in xylol (3 baths of 30 min each), and embedded in paraffin. Five‑micrometer-thick serial sections were obtained and stained with hematoxylin and eosin (HE). We used the HE‑stained sections to analyze the alterations in the arrangement of hepatic parenchyma and the presence Table 1 – Composition of the experimental diets Component (g/100g of diet) Diet 1 STD HLD HL/HCD Casein (82.93 g of protein) 16.47 16.47 16.47 L-cystine 0.18 0.18 0.18 Soybean oil 4.00 0.00 0.00 Lard 2 0.00 21.00 20.00 Cholesterol 2 0.00 0.00 1.00 Cholic acid 2 0.00 0.00 0.10 Sucrose 10.00 10.00 10.00 Cellulose 5.00 5.00 5.00 Mineral mix 3.50 3.50 3.50 Choline bitartrate 0.25 0.25 0.25 Vitamin mix 1.00 1.00 1.00 tert -Butylhydroquinone (TBHQ) 0.008 0.008 0.008 Dextrinized cornstarch 15.50 15.50 15.50 Corn starch 44.09 27.09 26.99 Lipids (g/100g) 3 4.1 ± 0.1 19.7 ± 1.4 20.8 ± 1.6 Energy from lipids 4 36 189 189 Diet energy (kcal/g) 4 3.41 4.26 4.26 (Energy from lipids x 100)/diet energy (%) 11 44 44 1 Reeves et al. 13 ; STD: standard diet; HLD: high-lard diet; HL/HCD: high-lard and high-cholesterol diet; 2 Fernandes et al. 10 ; 3 Result of diet chemical analysis; 4 Diet energy value: 4, 4, and 9 kcal/g for protein, carbohydrate, and lipid, respectively. 897