2.1. Study population
In this cross-sectional study, 218 out of 288 healthy women with overweight and obesity who were referred to community health centers of Tehran university of medical science were randomly selected based on the following inclusion criteria: 1) body mass index (BMI) 25-40 kg.m2 - 2) aged 18-50 years old. The primary exclusion criteria included the following:1) menopause 2) pregnancy 3) cardiovascular diseases 4) diabetes 5) Cancer 6) kidney disease 7) thyroid disease 8) acute or chronic diseases 9) use of dietary supplements for weight loss 10) follow up diet during the past year 11) use of lipid lowering drugs 12) use of blood glucose lowering drugs. More over, participants who did not complete more than 35 items of the food frequency questionnaire and those who reported total daily energy intakes ≤800 or ≥4500 kcal/d were excluded. This study carried out in accordance with the recommendations of the ethics committee of institution (Ethical number IR.TUMS.VCR.REC.1398.619 ) with written informed consent from all participants .
2.2. Anthropometric assessment
For each participant, the height, the weight, waist, and hip circumferences were measured. These measurements were made in compliance with WHO recommendations and the assessments were performed by an experience nutritionist. Participants were weighed without shoes and in minimal clothing on a solar digital scale recorded to the last 0.2 kg. The standing height was measured on a free-standing portable height meter with a precision of 0.5cm without shoes. BMI was calculated by weight (kg)/height (m) squared .
2.4. Dietary assessment and DTAC evaluation
Detailed dietary information was obtained through the use of the validated semi quantitative food frequency questionnaire (FFQ) . The FFQ includes 147 items with a standard serving size commonly consumed by Iranians. The reported frequency for each food item was then converted to a daily intake. The nutrient content of foods were computed by the Nutritionist 4 software based on United States Department of Agriculture (USDA) food composition table modified for Iranian foods. DTAC was calculated using the following indices: total reactive antioxidant potential (TRAP), Trolox equivalent antioxidant capacity (TEAC), and the ferric-reducing ability of plasma (FRAP).Since no databases were available to calculate the quantity of antioxidants in Iranian foods, given the available resources, we chose databases that contained most of the foods that are consumed by the Iranian population. TRAP and TEAC values were obtained from published databases for Italian foods. For FRAP, we used a database developed by Halvorsen et al. Total antioxidant capacity values for each food item in the FFQ were matched to an equivalent food in each of the databases. If any food was not directly matched with a corresponding food in a database, a proxy estimation was used based on the mean value of a similar food or the value of a raw food as a substitute for the cooked food [19, 21]. DTAC for every participant was obtained by multiplying the daily intake of each selected food item by its corresponding antioxidant value per food portion and summing the final values. Antioxidants from supplements were not included in the calculation of DTAC reported as micromole of Trolox Equivalents per day (μmol TE/day).
2.3. Biochemical analysis
Venous blood was drawn at 8:00 AM to avoid variations due to circadian rhythm and after a fast equal to or>12 h. Samples were collected in two tubes, one containing sodium citrate and the other without anticoagulant centrifuged at 3000 rpm for 15 min at 4 °C, and aliquots of plasma and serum were prepared for testing. Glucose, total cholesterol, HDL-C, and triglycerides levels were determined by the semiautomatic chemical analyzer Ekem KontroLab. LDL-C serum concentration was calculated with Fried Ewald’s formula. Insulin resistance was evaluated through homeostasis model assessment : HOMA-IR=insulin (mU/mL) x fasting glucose (mmol/L)/22.5.
2.4. Definition of metabolically healthy and unhealthy phenotypes
There are no general definition of metabolically healthy obesity (MHO).Throughout this paper, the MHO was described using adapted Karelis criteria. In the case of the Karelis criteria : total cholesterol ≤200 mg/dL, triglycerides≤150 mg/dL(≤1.7mmol.L), high-density lipoprotein cholesterol (HDL_C) ≥50 mg/dL and no treatment, low-density lipoprotein cholesterol (LDL-C) ≤100 mg/dL and no treatment, and HOMA-IR ≤2.8 ; that ≥4 positive score criteria defined as Metabolic Healthy. Thus, participants were classified into 4 groups according to metabolic health: Metabolically Healthy Over Weight (MHOW), Metabolically Unhealthy Over Weight (MUOW), Metabolically Healthy Obesity (MHO) and Metabolically Unhealthy Obesity (MUO) [8, 10].
2.5. The Genotype determination
The chromosome 9p21 rs17782313 SNP (genotypes C &T) was genotyped by the polymerase chain reaction-restriction fragment length polymorphism (PCRRFLP) technique. The Genomic DNA was extracted from 200 mL of whole blood using the Mini Columns Type G kit manual (GeneAll, South Korea). The extracted DNA was used to assess two reported SNPs near MC4R gene, rs17782313 and rs17700633 SNPs. Polymerase chain reaction (PCR) was performed on the rs17782313 and rs17700633 SNPs using the following primers: forward primer 5AAGTTCTACCTACCATGTTCTTGG-3 and reverse primer 5-TTCCCCCTGAAGCTTTTCTTGTCATTTTGAT-3. PCR was carried out on a total volume of 20 μL, containing 1 μl extracted DNA, 0.5 μl primers F, 0.5 μl primers R, 10 μl Permix (Amplicon, Germany), and 8 μl Distilled water, with the following conditions in a DNA thermocycler: 1- primary denaturation at 95°C for 2 min; 2- Thirty- five cycles of denaturation at 95°C for 30 seconds, annealing at 58°C for 30 seconds, extension at 72°C for 30 seconds; 3- final extension at 72°C for 5 min; 4- final step at 4°C. The amplification protocol consisted of a primary denaturation step at 94 ° C for 5 min, followed by 35 cycles of denaturation at 60 ° C for 1 min, annealing at 94 ° C for 45 s, and extension at 72 ° C for 1 min, and final extension at 72 ° C for 10 min. The digestion products were stained with ethidium bromide on a 2% agarose gel and imaged. 10% of the samples were directly sequenced for confirmation the PCR-RFLP results. The sequencing process performed using the ABI PRISM 3730 automated sequencer (Applied Biosystems, Foster City, CA, USA) . Amplified DNA (7 μl) was digested with 0.5 μl of BCII restriction enzyme (Fermentase, Germany) at 56 °C overnight. All products were visualized by agarose gel electrophoresis. Then, fragments containing three genotypes were distinguished: CC, CT, and TT.
2.6. Statistical analyses
Statistical analyses were performed using SPSS 20 (SPSS Inc., Chicago, IL, USA). Normal data distribution was determined using the Kolmogorov-Smirnov test. The ANOVA test and general linear model were used to assess differences in biochemical measurements and characteristics between groups. A general linear model adjusted for MC4R, DTACT as confounder effects was used. Coding of the SNPs was performed using an additive model. The study population characteristics are reported as mean ± standard deviation (SD). The Statistical significance was defined as p ≤ 0.05 for all analyses.