2.1 Design of intervention
The number of subjects who were participated in the study is based on the previous study in this area (Te Morenga, Levers, Williams, Brown, & Mann, 2011). This interventional non-randomized control trial included 60 overweight and obese females aged between (18-50) years for 6 weeks. Participants were recruited before obtaining informed consent and screened by the researchers to determine eligibility based on inclusion and exclusion criteria, and then informed consent was signed up from all the subjects who voluntarily agreed to participate in the study. Figure 1 summarized the groups of trials.
Group 1: IPWLM (n=31): included participants who followed the Ideal Protein Weight Loss Method (IPWLM) Diet selected from Jwel private Clinic. The disclosure statement of the clinic had been signed by the researchers.
Group 2: BD (n=29) were recruited from King Saud University campus to receive a balanced diet (BD).
2.2 Ideal protein weight loss method diet (IPWLM) group
At the baseline visit subjects were instructed about the diet and how to prepare ideal protein products. Also, were asked to choose as a minimum of three ideal protein foods for breakfast, lunch, dinner, and snack. Food products and supplements (multivitamins, omega3, calcium-magnesium, vitamin D, and potassium) were provided to them with instructions about the allowed food that will be consumed with products which include: 5-8oz of animal protein and 4 cups of low glycemic index vegetables. Supplementary File 1 shows the summary of the dietary recommendations in the IPWLM group.
2.3 Balanced diet (BD) group
At the baseline visit, all subjects were provided with information about my plate, food label, fat-free and low-calorie foods, calories and nutritional composition, health consequences of obesity, and related diseases. Supplementary File 2 shows a summary of the dietary recommendations in the BD group. Participants were undergone individual consultation during the intervention program, as well as printed dietary materials and handouts that were designed and provided to all participants to go home according to their assigned dietary regimen to facilitate the implementation of the dietary interventional program. Daily energy requirement (Kcal/day) was estimated using the Harris benedict equation based on age, gender, height, and weight.
The Food record was given to each subject to fill it daily for checking the compliance in the follow-up visit. It included the name and quantity of the food as well as ideal protein products eaten at each meal and snacks, supplements including (multivitamins, calcium, magnesium, zinc, potassium, omega-3, and vitamin D), salt intake, water intake, and exercise. As well as printed dietary materials and handouts that were designed and provided to all participants to go home according to their assigned dietary regimen to facilitate the implementation of the dietary interventional program. At the follow-up visit additional FFQ which included examining the intake around the two major food groups (snacks and main meals) was given to subjects. Food groups were defined according to products in the ideal protein website.
2.4 Primary Screening
Subjects were screened at King Saud University and Jwel clinic before participation in the study. Where the researcher completed a primary examination of anthropometric measurements and clinical interviews which included questions to determine eligibility based on inclusion and exclusion criteria, detailed health information obtained from all subjects using preset health questionnaire.
2.5 Exclusion & inclusion criteria
Patients with chronic diseases such as cancer, kidney, and liver disease, those on appetite, weight control, and psychology medications, anti-depressant drug and mood stabilizer, vegan, as well as those who participated in weight loss program from three to six months ago, pregnant and lactating, postmenopausal women and subjects under 18 years old, were excluded. All subjects were matched for age, BMI, as closely as possible. Only Saudi females, pre-menopausal, aged between (18-50) years and BMI ≥25 kg/ with normal ECG and blood pressure with no history of vascular disease, renal and liver disease were selected for the study. The infrastructure to undertake these studies forms part of an ongoing research program, and active facilities are available to facilitate this research. These studies were conducted for 6 weeks utilizing only women for this part of the study to increase the compliance rate. The flowchart describing the consort of participants through our clinical trial is presented in Figure 2.
2.6 Assessment component
After meeting inclusion and exclusion criteria participants were followed two different types of assigned weight loss methods. Participants were asked to come with overnight fasting (8-10hr) for undergoing a complete assessment. Anthropometric assessment, dietary assessment (food frequency questionnaire, food record), and biochemical assessment. Those measurements were obtained at baseline and at follow up visit after 6 weeks.
Participating subjects were requested to return to the clinic (at Jwel clinic or King Saud university clinic) for anthropometry. Anthropometric measurements were taken by a well-trained nurse. Anthropometric data were collected with an emphasis on clinical markers of adiposity. Seven variables were evaluated for anthropometric measurement: weight, height, body mass index, waist and hip circumferences, waist to hip ratio, and body composition.
Height was measured by stadiometer while subjects were standing on a flat horizontal surface with their back against the height board. Their heels, buttocks, and shoulder blades should touch the wall or surface of the height board. Before measurements, participants were asked to take a deep breath and hold until the headpiece is pressed against the head. The measurement was read at eye level, to the nearest 0.1 cm or 0.125 cm. Weight was obtained using the Platform scale. The scale was on a hard, flat, horizontal surface calibrated to zero before use. Participants were instructed to wear light clothing, stand over the center of the scale platform, facing front against the scale, without shoes, and with weight evenly distributed between both feet. Weight was determined to the nearest 0.2 Kg and the mean of two variables was taken. (NEBRASKA WIC. 2012)
Waist circumference and Hips were measured when zero ends of the measurement tape are held in one hand and the other end is held in the other hand. Areas have been measured were free of clothing or covered by as little clothing as possible. The tape was snug not tight enough to compress the soft tissue. Measurements were recorded to the nearest 0.1 cm. BMI was calculated as weight (Kg) / Height² (m²). Based on BMI, participants were classified as normal, overweight, or obese. Body Composition was assessed using the Tanita body analysis scale. Which gives the whole body composition measurements: weight, percentage of body fat, fat mass, fat-free mass, muscle mass, total body water (TBW), extracellular water (ECW), intracellular water, ECW/TBW ratio, body mass index, bone mass, physique rating, visceral fat rating, basal metabolic rate, and muscle mass balance. All measurements were repeated once post-intervention at 6 weeks.
2.8 Clinical intervention (dietary regimen)
For data collection, a pre-designed health questionnaire of Jwel private Clinic was used with some modifications. Some questions were modified, omitted, or added to the questionnaire taking into consideration the participant's understanding. The questionnaire was judged for its face validity through review by three experts in nutrition. The health questionnaire consists of socio-demographic data, medical history, daily habits, and dietary assessment. Participants were assessed two times over a period of 6 weeks. At baseline and follow up visit (after 6 weeks) anthropometric measurements were taken. Health questionnaire and food frequency questionnaires were obtained from subjects in each group at baseline and follow up visit (after 6 weeks) to determine the changes for 6 weeks. Pre-designed Food frequency questionnaire (FFQ) is the dietary tool that was used to assess the food intake of the subjects prior and post the dietary intervention (Al-Daghri et al., 2015; Al-Disi et al., 2010). The FFQ includes examining the intake around the five major food groups (starch, grains, meat & beans, dairy products, fruits, and vegetables) along with the consumption of the junk food which are defined as heavily processed, highly palatable, and hyper-energetic and are often deprived of the vitamins and essential nutrients found in whole unprocessed foods (Figure 3) since it poses the greatest risk to health and wellbeing (Blackburn, 2001); energy-dense, nutrient-poor products were also included illustrated through the consumption of fat (cream, ghee, and nuts), sweets (cakes, honey, jam, ice-creams, donuts, and chocolates), salty snacks (pop-corn), soft drinks and caffeinated drinks (coffee, black and green tea(. Also, contains Ideal protein meal replacement products.
The researcher interviewed the participants individually and the information was collected using FFQ to assess the qualitative and the quantitative aspects of the food consumed by the participants for 6 weeks to check for compliance. The health questionnaire was given again to all subjects to assess changes for 6 weeks.
All participants have received health advice according to their assigned type of diet and were interviewed individually. To support compliance health advice was confirmed by phone messages or E-mail (once/week), Participant was asked to record all food eaten daily for 6 weeks. Food records were collected by the researcher and returned with feedback. Food record for group IPWLM included the name and quantity of food, ideal protein products eaten at each meal and snacks, supplements including (multivitamins, calcium, magnesium, zinc, potassium, omega-3, and vitamin D), salt intake, water intake, and exercise. The food record for group BD included the quantity and component of food eaten in each meal and snacks, details about ready to eat products and drinks.
Nutrient intake was calculated using the USDA database (18th- 21st Ed, 2009, 2010, Program, ars.www.usda.gov), as for the Saudi Arabic traditional dishes were analyzed using the Arabic food analysis program (version 1, 2006). The evaluation of daily food intake was made by the means of total energy and the total nutrient intake, the percentage of the total calories derived from fat, protein, and carbohydrates. Dietary nutrients values were compared with dietary reference intake (DRI) for specified age and gender for macronutrients (carbohydrates and protein) as well as for micronutrients (DRI, 2008/2011, Supplementary file 3).
2.9 Blood sample collection
The blood samples were collected using sterile vacutainer blood collection apparatus. Green/Gray mottled Plasma Separator Tube (PST) with heparin was used. All samples were aliquot and stored in –20◦C freezer facilities in preparation for subsequent analysis by spectrophotometer. A spectrophotometer consists of two instruments, namely a spectrometer for producing light of any selected color (wavelength), and a photometer for measuring the intensity of light. The instruments are arranged so that liquid in a cuvette can be placed between the spectrometer beam and the photometer. The amount of light passing through the tube is measured by the photometer. The photometer delivers a voltage signal to a display device, normally a galvanometer. The signal changes as the amount of light absorbed by the liquid changes. If the development of color is linked to the concentration of a substance in solution, then that concentration can be measured by determining the extent of absorption of light at the appropriate wavelength.
2.10 Biochemical assessment
Blood samples from all subjects were analyzed for various biochemical parameters. blood glucose and lipid profiles (total cholesterol, HDL, and LDL) were determined using standard AMP diagnostic kits (Stattogger Strasse 31b 8045 Graz, Austria). The instrument was calibrated before analysis using quality control samples provided with the kits. The parameters were measured at baseline and follow up visits after 6 weeks for all groups.
2.11 Statistical analysis
Data were analyzed using (SPSS) version 22 (SPSS, Chicago, IL, USA). Power was used for the calculation of sample size ascertaining the difference between two dependent means (matched pairs). A final sample size of 60 with α = 0.05 and an effect size of 0.0.651 has a power of 0.200 to detect the difference. All variables are present as mean ± standard deviation. For comparison between baseline and post-intervention, a paired t-test has been used. For comparison between groups (IPWLM group, and BD group), a randomized sample of 15 subjects was selected from the BD group to minimize the variation in sample size between groups and an independent sample t-test has been used. Significance was set at p ˂ 0.05.