We have hypothesized that dietary macronutrient composition has different effects on the metabolic responses and arterial stiffness indices of normal and obese adults.
The study protocol is based on the SPIRIT guidelines (30). Figure 1 depicts the diagram of enrollment, intervention, and assessments. Table 1 shows the schedule of enrollment, intervention, and data collection for each participant. This is a parallel clinical trial, and the subjects have been consuming three different test meals (high-carbohydrate, high-fat, and high-protein) on different days with a one-week interval between each intervention.
Participants and Data Collection
Data collection has been performed by two nutritionists and one nurse, who have been trained on the proper use of various devices and are able to complete the questionnaires accurately. Since this is a pilot study, we cannot assess the attrition rate, and it is also not necessary to define and calculate the sample size.
Study Setting and Sample Population
This pilot, parallel clinical trial has been underway at Imam Reza Hospital of Mashhad, located in the northeast of Iran. The participants were recruited by local advertising with posters distributed at Mashhad University of Medical Sciences.
The inclusion criteria of the study are as follows: 1) age of 18-40 years; 2) adults with normal weight (body mass index [BMI]: 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) written informed consent to participate and 5) willingness to undertake the required fasting periods.
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 the metabolism (e.g., thyroid drugs, caffeine); 5) history of CVDs, hypertension, diabetes mellitus, hyperlipidemia, and neurological and/or neuropsychological disorders; 6) consumption of toxic substances; 7) use of supplements for weight loss or weight gain and 8) inability to partake in the intervention due to intolerance/dietary preferences.
Three interventions have been implemented in the current research, involving the consumption of high-fat, high-carbohydrate, and high-protein meals as the test meals in a randomized order. On the day of the intervention, the TEE of the subjects was determined via indirect calorimetry. The test meals contained 25% TEE for men, and 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 contained bread, butter, cream cheese, high-fat milk, and jam (50% fat, 10% protein, and 40% carbohydrates). The subjects would be asked to ingest the test meals within a maximum of 15 minutes on each test day.
Descriptive statistics will be used to compare and determine the primary characteristics of the study groups, and the normal distribution of the variables will be evaluated using the Kolmogorov-Smirnov test. In total, 20 subjects have been assigned to the obese and normal groups. The baseline comparisons between the groups will be performed using the independent samples t-test or Mann-Whitney U as appropriate.In addition, repeated measures analysis of variance (ANOVA) will be used to assess the timing effect of the research parameters in various phases of the study, as well as the possible overall significant differences of the parameters between the lean and obese men. In all the statistical analyses, the P-value of less than 0.05 will be considered significant.
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 intakes will be assessed using the valid and reliable food frequency questionnaire (31), 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 (32).
The level of physical activity in the participant will be evaluated using a validated questionnaire (33) and 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.
A screening questionnaire will be designed based on a researcher-made questionnaire and the available standardized questionnaires in order to acquire the 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.
At this stage, the participants will be asked to stay awake and motionless in the 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 hourly measurements for six hours after the consumption of the test meals. Furthermore, fasting REE will be measured in a quiet area at room temperature, with the participants in the supine position. The respiratory quotient will also be calculated based on the oxygen consumption and carbon dioxide production, and TEF will be measured as the difference of the postprandial subtracted by the fasting REE. The resting energy expenditure and substrate utilization will be also be estimated via indirect calorimetry after resting for 30 minutes.
Pulse Wave Analysis
All the patients will receive an ultrasound examination of the carotid artery in the supine position, with the head turned to 45° from the side to be scanned and the operator placed on their right side. In addition, brachial blood pressure evaluation and pulse wave analysis will be conducted in the supine position using the Sphygmocor XCEL device. Blood pressure will be measured after the 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 also be analyzed in accordance with the guidelines of the pulse wave analysis device manufacturer. After the measurement of the HR, cBP and AIx will be estimated using built-in algorithms.
Carotid-radial pulse wave velocity (PWVb) is a measure of arterial stiffness, which 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 in our study. 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 test meals.
Each test day will be implemented at 7 AM-2 PM; this 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 metabolic activity markers. 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.
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 requirements. 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) and after fasting from food, drinks (except water), caffeine, and alcohol for the past 12 hours.
Before the tests, all the subjects rested in the supine position for 30 minutes, lying on a bed in the semirecumbent position until the end of the tests. Initially, all the measurements will be performed in the fasting state and postprandially within six hours. At the next stage, the participants will randomly receive the test meals and given 15 minutes to take the meals under the supervision of the researcher until the test meal is completely consumed. Afterwards, the participants will complete a series of tests, including indirect calorimetry, PWV, and pulse wave analysis. In addition, blood samples will be collected within 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 would fast again, refraining from food and drinks (except water) for another six hours while the testing stage will continue. The testing conditions will be repeated on the next two test days. Figure 2 depicts the schedule of the study days.