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).