Study design
An 8-week parallel, randomized, double-blinded, placebo-controlled clinical trial was conducted to investigate the effect of Zn supplementation on atherogenic indices from September 2018 to September 2019 at the Motahari and Imam Reza Clinics, Shiraz, Iran. The study protocol was in accordance with the declaration of Helsinki and good clinical practice and adhered to CONSORT guidelines. It was also approved by the Ethics Committee of Shiraz University of Medical Sciences, Shiraz, Iran (IR.SUMS.REC.1397.105), and registered in the Iranian Registry of Clinical Trials (IRCT.ir; IRCT20191015045113N1; 08/12/2019). Written informed consent was obtained from participants at the beginning of the study.
Afterward, participants received lifestyle modification recommendations over a 2-week run-in period in which they were recommended to have at least 20 min of physical activity a day, eat 5 meals per day, replace simple carbohydrates with complex carbohydrates, replace refined grains with fruits and/or vegetables, and reduce consumption of high fructose corn syrup sweetened food. After completing the run-in period, participants were randomly allocated to the intervention or control groups by the researcher, using the simple random allocation method with the random table number, in order to receive a dietary plan, in addition to a Zn supplement or placebo in the intervention or control group for 8 weeks, respectively. Demographic, anthropometric, biochemical, dietary intake, and physical activity assessments were performed before and after the study (Fig. 1). Allocations were concealed in an opaque envelope until after the baseline assessments.
Study population
According to the previous study [9], the sample size was estimated at 25 in each group, to detect a 0.18 reduction in AIP score (SD = 0.2) with a significance level of 5% (α = 0.05), a test power (1 – β) of 80%, and a probability of 10% attrition rate. Inclusion criteria include overweight or obese patients aged 18–70 years with ultrasound-confirmed NAFLD, with no alcoholic beverage consumption, lack of viral hepatitis, liver cirrhosis, Wilson’s disease, acute fatty liver of pregnancy, hepatocellular carcinoma and a history of chronic liver disease, no lipodystrophy, lack of parenteral nutrition, not suffering from diseases that affect the bile and bile ducts, no severe weight loss during the previous 6 months, lack of congenital metabolic diseases, not taking drugs that cause fatty liver (methotrexate, tamoxifen, valproate, etc.), no pregnancy and lactation, lack of serum alanine transaminase level more than 10 times the allowable limit, no history of severe systemic diseases such as CVD and kidney disease, not having chemotherapy during the past year, no drug and alcohol poisoning, not taking any supplements containing Zn. Exclusion criteria included allergies and severe side effects from taking supplements, unwillingness to continue the study, failure to follow the recommendations and diet provided, and consumption of less than 90% of the supplements provided.
Intervention
The intervention group received one 30 mg Zn capsule (zinc gluconate, Nature Made, USA) daily and the control group received one placebo capsule (starch powder) daily with a meal for 8 weeks. Considering that the tolerable upper intake level (UL) of Zn in adults is 40 mg/day and the recommended dietary allowance (RDA) is 8-11 mg/day, in addition to the lack of any complications by the dose of 30 mg/day in previous studies [22, 23], this amount of Zn was considered as the intervention dose in this trial. Both groups also received a dietary plan with a caloric deficit of 500–1000 kcal/day, consisting of 50–55% carbohydrates, 30% fat, and 15–20% proteins, based on the participant’s estimated energy requirement.
Randomization and blinding
Randomization was performed using a simple random allocation method based on the published random table number by an independent statistician. The intervention and placebo capsules were similar in shape, color, and size and were put in similar containers coded as A or B by a person out of the study. Only the principal investigator (PI) could decode the contents of each capsule. The researchers in charge of capsule delivery and conducting the study, the patients, the physician, and the outcome assessor were blinded to the coding.
Compliance assessment
Telephone calls were made every two weeks to follow up with patients, record side effects, and prevent attrition rates. At the end of the fourth week, patients attended the clinic to receive the capsules. The number of capsules consumed was recorded. If the patients consumed more than 90% of the prescribed capsules, they were considered adherent.
Dietary intake assessment
A 24-hour food record for 3 days (2 regular days and a weekend day) was used to evaluate the dietary intake of participants. Then, food records were analyzed by a modified version of Nutritionist 4 software (First Databank Inc., San Bruno, CA, USA) for Iranian food items. Total energy, macronutrients, fiber, and Zn intakes were calculated.
Physical activity assessment
The International Physical Activity Questionnaire (IPAQ) was used to assess patients’ physical activity. This questionnaire consisted of 7 questions about the intensity and duration of physical activity during the past week. The metabolic equivalent (MET) for light, moderate and vigorous activities is considered 3.3, 4, and 8, respectively. Then the intensity (MET) of physical activity was multiplied by the duration (minute) of physical activity to calculate the amount of physical activity (MET*min/week).
Anthropometric assessment
Height was measured with a tape measure attached to the wall, in a standing position, and without shoes with an accuracy of 0.5 cm. The weight of patients in the lightest possible clothing without shoes was measured by a scale (Seca, Germany) with an accuracy of 100 g. Waist circumference was measured at the midpoint between the margin of the lowest palpable ribs and the upper edge of the pelvis with an inelastic meter parallel to the ground to the nearest 0.1 cm. Body mass index (BMI) was calculated based on the standard formula (weight (kg) / [height (m)]2).
Biochemical assessment
After 10–12 h of fasting, blood samples (5 cc) were taken from the subjects at the beginning and end of the study. Blood samples were centrifuged (4000 rpm for 10 min) and sera were stored in a freezer (-70 °C) until further analysis. Blood sampling was performed at the Motahari clinic laboratory, Shiraz, Iran. Then the samples were analyzed in the laboratory of the Faculty of Nutrition and Food Sciences of Shiraz University of Medical Sciences, Shiraz, Iran. Lipid profile (TC, TG, LDL-C, and HDL-C) levels were measured by the enzymatic photometric method by auto-analyzer (BT-1500, Italy (and commercial kits (Pars Azmoun, Iran). Serum Zn was measured by colorimetric spectrophotometry assay.
Atherogenic indices assessment
Atherogenic indices (AC, AIP, Castelli risk index I, and Castelli risk index II) were calculated using the following equations at the beginning and end of the study:
AC = (TC − HDL − C)/HDL– C.
AIP = log (TG/HDL − C).
Castelli risk index I = TC/HDL– C.
Castelli risk index II = LDL − C/HDL– C.
Statistical analysis
Data analysis was performed by Statistical Package for Social Sciences (SPSS) software (version 19.0, SPSS Inc., Chicago, IL, USA). P < 0.05 was considered significant. Data were reported as mean ± standard deviation (SD). The normality of data distribution was assessed using the Shapiro-Wilk test. The chi-square test was used to test the homogeneity of qualitative variables between groups. Within-group and between-group comparisons of variables were performed by Paired t-test and Independent Sample t-test for normally distributed data, respectively. ANCOVA (analysis of covariance) test was also used to adjust confounder variables.