2.1 Study design and patients
This triple-blind, placebo-controlled, randomized clinical trial was conducted between 2019 October and 2020 March. Seventy NAFLD patients (Body mass index (BMI): 25−35 kg/m2) aged 20-50 years old were engaged from Valiasr hospital of Tabriz, Iran and clinics via advertisements and posters. The subjects were diagnosed by a physician after an ultrasound examination on the basis of steatosis. Adherence to a stable diet, constant physical activity level (PAL) and willingness for participation were inclusion criteria of the study. The exclusion criteria were a history of biliary disease, hepatitis B and C, copper and iron storage disease; cardiovascular, gastrointestinal, and renal disease, pancreatic, thyroid disorders, cancer, smoking; pregnancy or lactation; post-menopausal; using antilipidemic and fatty liver inducing medications; currently antibiotics consumption, taking antacids, antidiarrheal, anti-inflammatory or laxative medicines and patients with special diets or dietary limitations. Also, patients were asked to consume minimum amount of nuts and fish and not to take antioxidants and omega-3 supplements. At baseline, patients were given a full information of the study and asked to sign a written informed consent. Also, they fulfilled a demographic questionnaire containing variables including the sex, age, PAL and the current medications of the subjects. The patients were requested to keep their usual PALs and follow the designed diet until the end of the intervention.
After a 2-week run-in period, patients were randomly allocated to either intervention (CSO, jahan, Tabriz, Iran) or placebo (sunflower oil (SFO), oila, Tehran, Iran) group based on age, sex, and BMI by block randomization method with a block size of 4. Random allocation software was performed to generate allocation sequence. The participants were assigned to the study groups by a third person (entirely unrelated to the trial) to achieve the blinding in the assessment process. The participants, principal researcher and statistical advisor were blind until the end of the analysis. The participants in the study groups were instructed to consume 15% of the daily total fat intake (~20g) from the provided oil during the intervention period. Measuring cups with the volume of 10 mL were provided for the subjects to add two cups of oil daily to rice or salad at the time of consumption. The rest of the required oil was provided from meats and low-fat dairy, as well as cooking oil. A calorie restricted diet was designed for participants in both groups (50-55% from carbohydrates, 30-35% from fat and 10-15% of energy from protein). After a full explanation of the diet, the patients received an exchange list to facilitate the adherence to the designed diet during the intervention. Also, patients in both groups were asked to consume the least amount of oil during cooking, low-fat dairy and meat and avoid fried foods. The half of the oil bottles was provided at baseline for participants and the remaining packages at 45 days of the study. Patients were followed up biweekly to remind the recommendations on oils, diet and PAL, in addition to explore any possible side effects. Also, a checklist was provided for participants to mark after each consumption of the prescribed oil to evaluate for cases of non-compliance.
The sample size was calculated regarding the changes in superoxide dismutase (SOD) level as one of the primary outcomes. A power study of 90% and a confidence interval of 95% 23 were assumed in the Pocock formula and calculated at least 21 subjects in each group. Also, considering a 10% attrition rate, the sample size increased to 23 per group.
The primary outcomes of the current study were high sensitivity- C reactive protein (hs-CRP) malondialdehde (MDA), 8-iso-prostaglandin F2α (8-iso-PGF2α), uric acid, SOD, total antioxidant capacity (TAC), glutathione peroxidase (GSH-Px), catalase and LPS. The secondary outcomes were insulin, and fasting plasma glucose (FPG).