Experimental protocol
The current research will be conducted in three phases:
Phase one: To explore the postprandial effects of five wheat bran bread products on individuals with T2DM.
Phase two: To evaluate the organoleptic characteristics of bread prepared with four doses of PPP.
Phase three: To determine the effects of consuming PPP-fortified bread on risk factors including obesity, oxidative stress, insulin resistance, inflammation factors, and lipid profile in type 2 diabetes patients.
Sample preparation
Pomegranates of a specific variety will be collected and purchased from cultivation areas in Neyriz, Fars Province, southwest of Iran. They will be washed with cold water and drained. They will be then cut open and the leathery skin on the outside will be removed. After that, the pomegranate and white parts of the interior pomegranate will be separated. The skin will be ground using an electric mill and sieved through a #12 mesh sieve. The powder will be stored in closed containers in a -20 °C freezer until use.
Organoleptic (sensory) tests
Different amounts of PPP will be added to wheat flour to obtain final percentages of 1.5%, 2.5%, 3.5%, and 5%. PPP-free samples will also be used as controls. The prepared traditional and bulk breads will be cut into equal-sized slices and served in unmarked dishes for 25 untrained panelists (university employees and students) to score their organoleptic properties on a five-point hedonic scale from one (extremely poor or lowest quality) to five (remarkably good or excellent quality). They will be ask to drink some water after testing each sample to cleanse their palate. We will also record and rate the internal and external characteristics of texture, color, aroma, taste, and mouth feel of the bread samples on a score sheet.
Determination of total phenol
Folin-Ciocalteu (FC) colorimetric method will be employed to determine the total phenolic content (TPC) of methanolic extracts of bread samples. Results will be expressed in milligrams of Gallic acid equivalents per gram of bread (mg GAE/g) (10, 14).
DPPH radical scavenging activity
DPPH will be used to assess the antioxidant activity of the extract. Using a spectrophotometer, the absorbance will be measured at 515 nm, and the percentage of DPPH scavenging activity will be calculated using the following equation (15):
Radical scavenging activity (%) = (Blank OD-sample OD/Blank OD) *100
The experiment will be performed three times and the average value will be reported. The amount of radical scavenging activity will then be calculated. Half-maximal inhibitory concentration (IC50) will be used to determine the substance’s ability to neutralize 50% of the initial free radicals in the environment. Phenolic and DPPH tests will be conducted to determine the phenol content of bread after adding PPP.
Setting
In the beginning, a list of outpatients with T2DM will be obtained from the Endocrine and Metabolism Research Center and various health centers in Isfahan, Iran. Study inclusion and exclusion criteria are in table 1.
Table 1: Eligibility criteria participation for the study
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Inclusion criteria
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Age between 40 and 60 years
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Informed consent
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HbA1c < 8
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Diagnosis of diabetes during the past five years
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BMI < 30 kg/m²
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Absence of complications
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Exclusion criteria
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Changes in diet or physical activity within the past six months
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Current smoking and/or alcohol or drug abuse history
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Pregnancy and lactation
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Renal failure (stage 3-5)
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Liver disease (cirrhosis and hepatitis)
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Insulin treatment or sulfonylureas
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Current involvement in a clinical trial
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Recent weight change
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Use of non-steroidal anti-inflammatory drugs
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History of malignancy
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Being on a special diet (e.g., vegetarian and ketogenic)
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Weight loss over 10% in the past six months
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Volunteer patients will be invited to contact the research team by phone, attend an examination, and complete a brief questionnaire about their health history. The study protocol will be explained to the participants and they will be asked not to make any changes to their usual diet during the study. Groups 1 and 2 will receive wheat bread (100 g) named A and B, respectively. Both the researchers and the patients will be blinded to the randomization code of the intervention and placebo breads (A and B).
Run-in period
The study will last for three months, beginning with a 14-day run-in period. The participants will be asked to maintain their dietary habits, medicines, and exercise. The run-in period will help the researchers to become familiar with the patients’ diets and to identify their concerns. During the run-in period, the participants will be consuming regular wheat bread. The run-in phase is necessary to test the volunteers’ motivation to comply with the intervention. Since there is no biomarker to measure bread intake, we will use diaries to assess their compliance.
Study design
The study will be conducted on 90 diabetic patients referred to Isfahan Endocrine and Metabolism Research Center and other health centers in the city. Patients will be given the reasons for the work and how the plan is implemented. A questionnaire containing demographic data, age, gender, telephone number, cell phone, address, and medical records will be used to evaluate the inclusion criteria. Individuals who wish to participate in the study will be asked to sign an informed consent form after the approval of the study. The participants will be assigned to either the intervention or control group (Figure 1) and both groups will continue their routine treatment plans. In addition to their routine diet, the participants in the intervention group will also have to eat 100 g regular bread every day. Variables will be assessed at baseline and at the end of the trial.
Informed consent form process
Informed consent will be obtained from all participants. An approval from the Medical Ethics Committee of Isfahan University of Medical Sciences will also be needed. Before their enrollment, the patients will be assured that their unwillingness to participate will not affect their usual health care at health centers. The participants will have enough time to review the consent form and ask any questions they should have. They will also receive a copy of the consent document after signing it.
Anthropometric Assessment
Anthropometric parameters
We will measure height, weight, and waist and hip circumferences at baseline and three months post-intervention. Height and weight (with a precision of 0.01 kg) using a scale-mounted stadiometer while the patients wear light clothing and no shoes. Each participant’s BMI will be calculated as the result of dividing their weight (in kg) by their height (in m) squared. A flexible tape will be used to measure waist and hip circumference to the nearest 0.1 cm. Waist circumference will be measured at the midpoint between the lowest rib and the iliac crest while the patient’s body is level and they are wearing minimal clothing. Hip circumference will be measured around the widest portion of the buttocks. Only one person will do measurements to reduce error rates.
Physical activity and dietary intake evaluation
At the beginning of the intervention and 12 weeks later, food and activity records will be assessed. For the purpose of dietary intake evaluation, the participants will be asked to record their daily food and beverage intake for three days. We will assess dietary intake using the food records of three nonconsecutive days (two weekdays and a weekend). A food scale and a model will also be used to improve accuracy. The three-day food record will be reviewed by the investigator in the presence of the patient. We will convert the portion sizes to grams. Using Nutritionist 4 (First data bank Inc., Hearst Corp., San Bruno, CA), each food and beverage item will be coded and analyzed for energy content and macro- and micronutrients. Individuals consuming less than 1200 kcal/day or more than 4000 kcal/day will be excluded The Participants will complete the Metabolic Equivalent Questionnaire (MET) for physical activity before and after the intervention and will also be asked to report their physical activity on a daily basis. They will receive bread packages twice a week during the 12-week intervention period. As there is no biomarker for assessing the consumption of PPP-fortified bread, compliance with the research protocol (i.e., daily intake of bread) will be evaluated using a self-reported questionnaire.
Sample size calculation
The sample size was calculated by considering the first type error (α=0.05), the second type error (β=0.2), SD= 32 and 42, and the minimum significant difference for cholesterol (23mg/dl) as the main outcome variable.
The sample size for each group was 40 participants. Including a 10% chance of dropping out, 45 people were determined. Sampling is expected to last for 14 months. The participants’ data will be recorded even if they withdraw from the program (16).
Blood pressure
Blood pressure will be measured after 10 minutes of resting in a seated position using a mercury barometer calibrated in that position. The patients will be asked not to drink tea/coffee or engage in heavy physical activity for approximately 30 minutes before the measurement. At the beginning and end of the study, blood pressure measurements will be performed twice and the mean of two consecutive readings will be recorded.
Biochemical assays
In order for laboratory tests, patients who meet the inclusion criteria (Table 1) will be asked to attend Isfahan Endocrine and Metabolism Research Center between 8:00 am and 10:00 am after fasting for 12 hours. During their appointment, 10 cc blood samples will be obtained. The collected samples will be centrifuged at 3500 rpm, 25 ℃ for 15 minutes. The serum will then be transferred and stored in a -80 ℃ freezer until further analysis. The participants will be tested for their biochemical parameters at baseline and after the intervention. Homeostatic model assessment of insulin resistance (HOMA-IR) and homeostatic model assessment of beta-cell function (HOMA-B) index will be used to determine insulin resistance (primary outcome) and beta-cell function, respectively. To determine insulin sensitivity, check index (Quick) will be measured based on the following equation:
HOMA-IR= (glucose-insulin)/405; Where glucose is fasting glucose (mg/dl) and insulin is fasting insulin (mu/ml) (17-19).
Statistical analyses
SPSS software will be used for the statistical analysis of data. Data will be expressed as mean ± SD for continuous variables and frequency report for qualitative variables. Kolmogorov-Smirnov test will be used to determine the compliance of the data with the normal distribution. Paired samples t-test will be used to compare baseline data between and within the two study groups for normal data. Paired samples t-test will be used to compare baseline data between and within the two study groups for normal data and Manwitny test for non-normal data. The baseline demographics as well as clinical and diabetes-related Characteristics of the intervention and the control groups will be presented and compared. The average changes between baseline and 3 months in primary and secondary outcomes will be calculated for each of the groups. Intention-to-treat (ITT) analysis will be performed as the primary analysis on all primary and secondary outcomes after the last participant has ended participation. Missing values will be handled with the last observation carried forward approach for ITT analysis with the use of the multiple imputation approach in a sensitivity analysis.
Two-sided tests will be used. Also, in all analyzes, the value of P-value <0.05 will be considered statistically significant.
ETHICS AND dissemination
Individuals provided written informed consent the study will be conducted in compliance with the Declaration of Helsinki. The trial has received ethical approval from the Ethics committee of Isfahan University of Medical Sciences (number:IR.MUI.RESEARCH.REC.1399.087). The study results will be disseminated through peer-reviewed publications and presentations at scientific meetings.
Study outcomes
The primary outcome of this study is total Cholesterol. Secondary outcomes are insulin resistance and beta-cell function measured by the homeostasis model of assessments (HOMA) and quantitative insulin sensitivity check index (QUICKI). Further secondary outcomes include weight, waist circumference, blood pressure, lipid profile, hs-CRP, and oxidative stress biomarkers such as MDA and TAC.