Research Hypothesis
Dietary macronutrient composition has different effects on the metabolic responses and arterial stiffness indices between normal and obese adults.
Experimental Design
The study protocol was based on the SPIRIT guidelines (29). The diagram of the enrolment, intervention, and assessments in the experiment is depicted in Figure 1. Table 1 shows the schedule of enrolment, intervention, and data collection in each participant. This will a parallel clinical trial, in which each subject will be consumed three different test meals (high-carbohydrate, high-fat, and high-protein) on various days with the washout period of seven days between each intervention.
Table 1.
SPIRIT 2013 figure showing schedule of enrolment, intervention and data collection for each participant. * randomly assigned meal.
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STUDY PERIOD
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Enrollment
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Allocation
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Post-allocation
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Timepoint
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preintervention
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Time 0
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baseline
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Intervention Visit 1
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Intervention Visit 2
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Intervention Visit 3
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Enrolment
Eligibility screen
Informed consent
Allocation
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Interventions
High protein
High charbohydrate
High fat
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Data collection
Food frequency
Questionnaire
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International physical activity questionnaires
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Anthropometric variable
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FAT and Lean mass
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Energy expenditure
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Resting metabolic rate
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Charbohydrate oxidation
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Fat oxidation
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Protein oxidation
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Blood pressure
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Heart rate
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Augmentation index
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Pulse wave velocity
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Serum glucose and insulin levels
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Lipid profile
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Participants and Data Collection
Data collection will be performed by two nutritionists and one nurse, who will be trained on the proper use of various devices and will be able to complete the questionnaires accurately. Since this is a pilot study, we could not assess the attrition rate, and it is not necessary to define and calculate the sample size.
Study Setting and Sample Population
This pilot parallel clinical trial will be conducted at Imam Reza Hospital in Mashhad, located in the northeast of Iran. The participants will be recruited by local advertising with posters at Mashhad University of Medical Sciences.
Inclusion Criteria
The inclusion criteria of the study are as follows: 1) age of 18-40 years; 2) adults with normal weight (body mass index [BMI] of 18.5-23.5 kg/m2, body fat percentage: 12-22%, waist circumference<90 cm) and obese adults (BMI>27.5 kg/m2, body fat percentage>27%, waist circumference>95cm); 3) apparently healthy men; 4) providing written informed consent and 5) willingness to undertake the required fasting periods.
Exclusion Criteria
The exclusion criteria of the study are as follows: 1) professional athletes; 2) greater changes in the body weight than 10% within the past six months; 3) current smoking habits; 4) use of medications or supplements affecting metabolism (e.g., thyroid drugs, caffeine); 5) history of CVDs, hypertension, diabetes mellitus, hyperlipidaemia, neurological, and/or neuropsychological disorders; 6) consumption of toxic substances; 7) use of supplements for weight loss and weight gain and 8) subjects unable to partake in the intervention due to intolerance/dietary preferences.
Test Meals
Three interventions will be implemented in the current research involving the consumption of high-fat, high-carbohydrate, and high-protein meals as the tests in a randomized order. On the day of the intervention, the TEE of the subjects will be determined using indirect calorimetry. The test meals contained 25% of TEE for men. The high-protein meal consisted of bread, cheese, boiled eggs, and skimmed milk (30% protein, 50% carbohydrates, and 20% fat). The high-carbohydrate meal consisted of skimmed milk, white bread, butter, jam, and honey (70% carbohydrates, 20% fat, and 10% protein). The high-fat meal consisted of bread, butter, cream cheese, high-fat milk, and jam (50% fat, 10% protein, and 40% carbohydrates). The subjects will be ingested the test meals within a maximum of 15 minutes on each test day.
Statistical Analysis
Descriptive statistics will be used to compare and determine the primary characteristics of the subjects in the study groups, and the normal distribution of the variables will be evaluated using the Kolmogorov-Smirnov test. In total, 20 subjects will be assigned to the obese and normal groups. Independent-samples t-test and Chi-square will be applied for the variables with normal distribution and inter-group comparisons. Moreover, paired sample t-test and McNemar test will be used for the intra-group comparisons. In the case of the data with non-normal distribution, the Kruskal-Wallis test will be used for the inter-group comparisons, and the Wilcoxon test will be employed for the intra-group comparisons. In all the statistical analyses, the P-value of less than 0.05 will be considered significant.
Measurement Tools
Anthropometric Parameters and Body Composition
The anthropometric measurements will be performed at the outset of the fasting state by a trained nutritionist. In addition, body weight will be measured to the nearest 0.1 kilogram, with the participants in light clothing. The height of the subjects will be measured using a stadiometer in the standing position to the nearest 0.1 centimeter. BMI was defined as weight (kg) divided by the square of height (m2), and waist circumference will be measured at the midline between the iliac crest and lowest ribs to the nearest 0.5 centimeter. Finally, the body composition of the participants will be determined using bioelectrical impedance analysis (AVIS 333).
Dietary Measurements
Dietary intakes will be assessed using the valid and reliable food frequency questionnaire (30), and the collected data will be expressed as gram per day using household measures. In addition, the modified food consumption database of the US Department of Agriculture will be used to calculate the daily nutrient intake of each subject (31).
Physical Activity
The level of physical activity in the participant will be evaluated using a validated questionnaire (32), calculated based on the metabolic equivalent tasks. Based on the questionnaire, the participants will be divided into three groups of low, medium, and high physical activity.
Screening Questionnaires
A screening questionnaire will be designed based on a researcher-made questionnaire and the available standardized questionnaires in order to acquire general data of the participants. Accordingly, the subjects will be excluded for medical reasons such as hypertension, CVDs, hypothyroidism/hyperthyroidism, intolerance, allergies, diabetes mellitus, and other acute/chronic diseases.
Indirect Calorimetry
At this stage, the participants will be asked to stay awake and motionless in a supine position, and air samples will be collected using a mask (MetaLyzer 3B-R3 device). Moreover, respiratory gas exchange measurements will be recorded for 20 minutes in the fasting state, followed by every hour for six hours after the consumption of the test meals. Fasting REE will be measured in a quiet area at room temperature, with the participant in a supine position. In addition, the respiratory quotient will be calculated based on the oxygen consumption and carbon dioxide production, and TEF will be calculated as the difference of the postprandial minus the fasting REE. The resting energy expenditure and substrate utilization will be also measured via indirect calorimetry after the participants rested for 30 minutes.
Pulse Wave Analysis
All the patients will be received an ultrasound examination of the carotid artery in a supine position, with the head turned to 45° from the side to be scanned and the operator on the right side of the patient. In addition, brachial blood pressure evaluation and pulse wave analysis will be conducted in a supine position using the Sphygmocor XCEL device. Blood pressure will be measured after a minimum rest of 15 minutes on the right upper arm in a quiet area. The augmentation index (AIx), central blood pressure (cBP), and heart rate (HR) will be also analyzed in accordance with the guidelines of the manufacturers for pulse wave analysis. HR will be measured, and cBP and AIx were estimated using built-in algorithms.
Carotid-radial PWV
Carotid-radial pulse wave velocity (PWVb), which is a measure of arterial stiffness, will be determined based on the sequentially measured electrocardiogram-gated left carotid and radial waveforms (applanation tonometry) using the foot-to-foot method to determine the pulse travel time. Moreover, the travel distance of the pulse wave will be calculated as the difference in the distance between the suprasternal notch and each recording site using a tape measure over the body surface. The measurements will be performed at least twice, and the mean PWVb will be applied to the analysis. PWVb will be assessed at baseline, and 30, 90, 150, 210, 270, and 330 minutes after the meals.
Blood Samples
Each test day will be implemented during 7 A.M-2 P.M, and the period was selected since it was assumed to represent a common interval between breakfast and lunch. Serum samples will be collected before meal consumption and 60, 180, and 360 minutes after the meal initiation to evaluate the markers of metabolic activity. A maximum of three milliliters of blood will be collected from each patient at each of the given times (12 ml/day) for the analysis of insulin, glucose, FFAs, TG, low-density lipoprotein, high-density lipoprotein, and total cholesterol. The blood samples will be collected at room temperature and immediately centrifuged, and the serum samples will be frozen at the temperature of -20°C.
Experimental Protocol
The day before each of the three test days, the subjects prepared their own meals and a standardized daily menu plan, which consisted of 15% protein, 55% carbohydrates, and 30% fat based on their daily energy requirement. The diets ensured equally filled glycogen stores and similar macronutrient balance in each subject on every test day. The subjects will not be allowed to engage in severe physical activity within two days prior to the test days. On the test day, the subjects arrive at the research center at Imam Reza Hospital at 7 AM with minimum activity (by car/bus) after fasting from food, drinks (except water), caffeine, and alcohol for the past 12 hours.
Before the tests, all the subjects rested in a supine position for 30 minutes, lying on a bed in a semirecumbent position until the end of the tests. Initially, all the measurements will be performed in the fasting state and postprandially during six hours. Following that, the participants will have 15 minutes to take their test meals under the supervision of the researcher until the test meal is completely consume. At the next stage, the participants will be completed a series of tests, including indirect calorimetry, PWV, and pulse wave analysis. In addition, blood samples will be collected during six hours. All the tests will be carried out in the exact same conditions (e.g., temperature-regulated room and quiet area). Following the test meals, the participants fasted again, refraining from food and drinks (except water) for another six hours while the testing stage will be conducted. These conditions will be repeated on the next two test days. The scheduling of the study days is depicted in Figure 2.