Design and setting
We will be performed a 2 month’s double-blind, parallel-group, clinical RCT. The proposed RCT will be conducted at the Nutritional research centre, Department of Nutrition, Ahvaz Jundishapur University of Medical Sciences for 2 months to evaluate the effect of daily 3 gr Tau supplementation in obese individuals. Fig 1.
Aims and study hypotheses
The primary aim of the present RCT is the assessment of 2 month’s Tau supplementation on serum levels of fibroblast growth factors (FGF19, FGF21), β-Klotho co-receptor, glycemic status, lipid profile, adipocytokines, hs-CRP and body composition in obese women on a weight-loss diet. Furthermore, the secondary aim will evaluate the associations between changes in concentrations of fibroblast growth factors (FGF19, FGF21) and β-Klotho co-receptor with other variables.
Participants will be 50 obese women aged between 18 to 50. Inclusion criteria will be consisted: women18 to 50 years old; body mass index (BMI) range between 30 to 40 kg/m2; absence of menopause, pregnancy and lactation; having no history of food allergy, cancer, acute or chronic renal failure, acute or chronic hepatic failure, thyroid disorders, gastrointestinal diseases, surgery for weight loss and any weight loss over the past six months; no taking Multivitamin/mineral supplements, herbal supplements or weight-loss drugs. Exclusion criteria will be included: pregnancy during the intervention, unwillingness to continue, no taking Tau supplement less than 90% of total prescribed capsules at the end of the study.
Sample size and sampling
The sample size was calculated based on the effect of Tau supplementation on changes in hs-CRP that was conducted by Rosa et al.. It was computed by considering 95% confidence interval and 80% power (α = 0.05 and β = 0.2). In addition, the mean and SD of hs-CRP levels in the mentioned study was as follow: µ1=14.30; µ2= 10.50; SD1= 2.90; SD2= 2.40. Finally, 25 subjects considered for each group. To reach the target sample size, all individuals who meet the inclusion criteria will be included in the RCT if they will be willing to participate in the study.
Randomization and blinding
Eligible subjects will be divided based on age randomly into two groups including control (standard weight loss group + taking placebo, n=25) and intervention (standard weight loss group + Tau supplementation, n= 25). Randomization will be performed using the computer-generated random numbers. The naming of Tau or placebo bottles will be done based on random numbers and odd or even numbers will be allocated to the A or B group. To achieve allocation concealment, the bottles will be sealed and we will be assured from the similarity of appearance and their weight. Randomization will be performed by persons other than the study investigator to reduce the probable bias. The researcher and participants will be blinded to the treatment allocation. Collected data will be held confidential. Randomization codes of RCT will be unlocked only after all individuals complete the study protocol.
All subjects will follow a hypocaloric diet, which energy needs will calculate by Mifflin Jeor St equation. Then, 30% of estimated energy requirements will deduct. The intervention group will receive 1 gr Tau capsule three times a day after breakfast, lunch and dinner. The Tau supplementation dose will be determined according to the previous study .Tau supplement will provide by Nutricost Company (USA). In addition, Placebo capsules will provide by the Pharmacy Faculty of Ahvaz Jundishapur University of Medical Sciences in the same form and size of Tau capsules. All capsules will be given to participants in the similar packing every 15 days. The macronutrients of a hypocaloric diet will be 50% carbohydrate 30% fat and 20% protein. Considering the general principles of diet, a trained dietician will give a dietary exchange list and an individualized diet according to the subjects’ dietary habits. The same dietician will follow subjects to check compliance through phone calls or SMS every three days. Figure 2 shows schedule for enrolment, intervention and assessment based on SPIRIT.
An individual information questionnaire including demographic situations, history of diseases, supplementations and medications will be filled at the baseline. Dietary intake will be evaluate by 3 days 24-hour recall quistionaires (2 weekdays and 1 weekend day) at the beginning, middle and the end of study. Total calorie and macroniutrients intake will be calculated using Nut IV (the Hearst Corporation, San Bruno, CA). The participants will be asked not to change their physical activity level (PAL). Physical activity levels will be assessed by the International Physical Activity Questionnaire (IPAQ) at the beginning and end of the study. The physical Anthropometric measurements will be given with minimal clothing and without shoes. Body weight will be measured using a 100 g accuracy scale (Seca). Height will be measured using a 0.5 cm accuracy Seca stadiometer. BMI will be computed by dividing body weight (kg) to the square of height (m). Waist circumference will be measured using tape meter at the midpoint between the lowest rib and iliac crest and at the end of normal expiration to the nearest 0.5 cm. TANITA BC-418 body analyser will be applied to estimate total body fat, fat percent, fat-free mass and fat free mass percent. 5 mL of venous blood sample (in regular tube) will be taken after 10-12 h overnight fasting at the baseline and end of study. Blood samples will be centrifuged at 1500 g for 15-20 min to separate serum. Serum will be stored at -80 °C and will be used to measure biochemical analyses such as FGF19 (μg/mL) , FGF21 (μg/mL), β-klotho co-receptor (μg/mL), leptin (μg/mL), adiponectin (μg/mL), hs-CRP (μg/mL), insulin (μU/mL), fasting blood sugar (FBS) (mg/dL), total cholesterol (TC), high-density lipoprotein (HDL-C), triglyceride (TG), alanine transferase (ALT), aspartate transaminase (AST) and gamma-glutamyl transferase (GGT) in serum. Enzyme-linked immunosorbent assay (ELISA) kits will be applied to measure serum FGFs, β-klotho co-receptor, leptin, adiponectin, hs-CRP and insulin. Lipid profile, serum glucose concentration and serum hepatic enzymes will be measured using enzymatic method by pars-azmoon kits (Tehran, Iran). Low-density lipoprotein (LDL-C) concentrations will be computed by the Friedewald equation. Homeostasis model assessment – insulin resistance (HOMA-IR) will be calculated as follow: FBS (mg/dL) × fasting serum insulin (μU/mL)/405.
We will be used intention to treat (ITT) and pre-protocol (PP) populations in the analysis. The ITT population consists all individuals who will be randomized, whereas the PP population consists all participants who complete the 8-week intervention. The data will be revised randomly to check accuracy and completeness. All data will be reported as mean ± SD. The changes percent for each variable will be computed by the following formula: [(E – B) / B × 100], where E and B are the end value and the baseline value of variable, respectively. The data normality will be analysed using Kolmogorov-Smirnov test. To compare parametric continuous data between and within the groups, will be used independent sample t-test and the paired sample t-test, respectively. In addition, to compare the differences in asymmetric variables between and within the groups, will be applied Mann-Whitney test and Wilcoxon test, respectively. The analysis of covariance (ANCOVA) test will be used to control confounding variables. SPSS version 21 (IBM, Armonk, NY, USA) will be used to data analysis. The p value < 0.05 will be considered statistically significant.
Safety, adverse effects and monitoring data
There are no side effects for 3 gr/day Tau supplementation . However, this RCT will supervise by a Data Monitoring Committee (DMC). In addition, any possible side effects will be reported to the Ethics Committee of the Ahvaz University of Medical Sciences. Moreover, the compliance and possible side effects such as digestive problems will be assessed by meetings, SMS or phone calls.