Study design and centers
The GENUTRI study is a single-center, randomized, open-label, parallel-group, 12-week pragmatic clinical trial (blinded outcome assessment). The study population will include patients with the diagnosis of CAD identified during a period of hospitalization in the Institute of Cardiology of Rio Grande do Sul (IC/FUC), Brazil, and outpatients who volunteered for the trial. The randomization, allocation and follow-up procedures will take place at the outpatient Clinic of Nutrition.
Study objectives
The primary objective of this randomized-controlled clinical trial is to evaluate the effect of three dietary approaches on metabolic, inflammatory and anthropometric profiles in patients with CAD after 12 weeks. The secondary objective is to detect an interaction of patients’ response to these three dietary approaches with polymorphisms in the CD36 and STAT3 genes.
Participants
Inclusion criteria: patients aged 40–80 years diagnosed with CAD who have signed an informed consent to participate. CAD was defined by a diagnosis of previous myocardial infarction, acute coronary syndrome or typical angina pectoris. Myocardial infarction was defined according to the universal definition of myocardial infarction [19].
Exclusion criteria: psychiatric disease; morbid obesity (body mass index (BMI) ≥40 mg/m2); expectancy of life less than 6 months; pregnancy or lactation; renal failure (in dialysis); congestive heart failure; prior organ transplantation; uncontrolled hypo/hyperthyroidism; wheelchair-dependant individuals; use of vitamin/nutritional supplements; chronic use of nonsteroidal anti-inflammatory drugs; and concomitant participation in another experimental study.
Ethical aspects
All procedures will be conducted in accordance with the ethical standards for human subject research set forth in the Declaration of Helsinki and also with the Good Clinical Practice (GCP) guidelines [20]. Written informed consent will be obtained by researchers from all patients included in the trial. The project was approved by the Research Ethics Committee of the Institute of Cardiology (registration number UP. 4861.13) and is registered in the ClinicalTrials.gov database.
Study protocol
Patients with CAD who have been hospitalized in our institution and/or those submitted to coronary interventions for CAD will be invited by telephone calls to participate in the study. For the individuals who meet the inclusion criteria and are eligible for the trial, an appointment will be scheduled with the researchers’ staff (physicians and nutritionists). Voluntary individuals who contact the investigators and who meet the inclusion criteria will be also scheduled. All individuals will be advised by telephone to fast overnight for 8 hours before the meeting.
After checking all exclusion criteria and signing the informed consent, a blood sample will be collected and the subjects will undergo a baseline cardiology assessment. A structured questionnaire containing demographic, socioeconomic and educational data will be administered. Medical history (including previous outcomes and drug prescriptions) and behavior data, such as smoking, alcohol intake and level of physical activity will be collected. Blood pressure levels will be evaluated; anthropometric data (weigh, height, waist, hip and neck circumferences) and records about actual dietetic consumption will be assessed.
After randomization and once allocation has been completed, the patient will be appropriately advised according to the intervention designated: pecan nut supplementation diet, olive oil supplementation diet or a control diet. Patients will be followed for a period of 3 months (12 weeks), and follow-up visits will be scheduled at 30 days, 60 days and 90 days (final appointment), when blood will be collected for laboratory analyses. Figure 1 shows all procedures and its periodicity during the trial according to follow-up visits. The patients will be reminded and prompted regarding the follow-up visits and the interventions by telephone calls.
Randomization and blinding
The randomization sequence (in blocks) will be computer-generated using the http://www.randomization.com/ website, and then organized in sealed opaque envelopes. After fulfilling the eligibility criteria during the first visit, participants will be randomly assigned by independent investigators to one of the three diet groups in accordance with the generated randomization list. Both patients and investigators responsible for evaluation and data collection will be aware of the assigned diet. The staff involved in the biochemical outcomes’ evaluation and in the statistical analysis will be blinded to group allocation.
Interventions
All individuals will receive individual dietary advice according to their specific caloric needs (to maintain or to lose weight) despite treatment group, and the following recommendations will be used to calculate the total daily calories: to weight management: 25 kcal/kg/day; to weight loss: 20 kcal/kg/day [21]. The individual distribution of macronutrients (dietary carbohydrates, protein and fats), independent of the group allocated, will be determined and calculated according to Brazilian guidelines for dyslipidemia [22], metabolic syndrome [21], hypertension [23] and diabetes [24].
Group 1 (nut supplementation, NS) will receive, in addition to dietary prescription, 1 kg of pecan nuts adequately packed for 30 days of treatment (approximately 900 g, considering an intake of 30 g/day [25]) plus educational materials concerning the adequate ingestion and storage of the nuts. Patients will be counseled about not consuming olive oil during the treatment. Group 2 (olive oil supplementation, OS) will receive, in addition to dietary prescription, 1 liter of extra-virgin olive oil adequately packed for 30 days of treatment (approximately 900 ml, considering an intake of 30 ml/day [25]) plus educational materials concerning the adequate ingestion and maintenance of the olive oil. Patients will be advised to not consume mixed nuts and oilseeds during the study period. Group 3 (control diet, CD) will receive standard dietary advice according to Brazilian guidelines [21–24] and will be discouraged from using olive oil, nuts and oilseeds during this period.
Assessment tools and variables
Demographic variables
A structured questionnaire will be administered to all participants for collection of demographic parameters (age, sex, ethnicity), socioeconomic and educational data.
Genetic data
Deoxyribonucleic acid (DNA) will be isolated from a 5-ml venous blood sample by means of a specific toolkit according to the manufacturer’s instructions. The detection of polymorphisms and genotype characterization will be performed from frozen blood samples at −80 °C by means of the TaqMan® SNP Genotyping Assay according to the manufacturer’s instructions and to the available protocol. For each gene, a different genotyping assay kit will be used: ID C_30301828_10 to detect the G allele in rs8069645 STAT3 and ID C_ 8314999_10 to detect the A allele in rs1761667 CD36.
Clinical and behavioral variables
Medical history: data on CAD specifications, history of present illness, past medical history, history of previous outcomes (i.e., heart attack and stroke) and current medications will be collected during the patient interview. Hypertension, type-2 diabetes mellitus and dyslipidemia will be considered present according to previous diagnosis and/or the use of medications to treat each of these conditions.
Blood pressure: systolic and diastolic blood pressure will be evaluated using an automatic validated monitoring device (OMRON® HEM-7200) with an adequately sized cuff to the arm circumference, according to the guidelines [23].
Body weight and height: weight (kg) will be measured with participants barefoot, wearing minimal clothing, and standing at the center of a digital platform scale; height (cm) will be obtained with participants barefoot in the standing position, and with both arms hanging freely at the side with palms facing thighs.
Waist, hip and neck circumferences: waist circumference (cm) will be obtained with a plastic, flexible measuring tape at the middle point between the lower costal margin and the iliac crest in a perpendicular plane, with the patient standing on both feet, approximately 20 cm apart, and with both arms hanging freely. Hip circumference will be measured at the level of the widest circumference over the buttocks with the research assistant kneeling at the side of the participant, so that the level of maximum extension can be seen. Neck circumference (cm) will be measured with the head straight and eyes staring forward, horizontally, 1 inch above the laryngeal prominence.
Smoking and alcohol intake: smoking will be defined as a categorical variable (never, current or past smoking) and excessive alcohol intake will be detected if participants have a consumption of 30 g or more and 15 g or more of ethanol a day for men and women, respectively [26].
Dietary evaluation: food intake will be evaluated by means of 24-h dietary recalls (24-HDRs). Caloric intake and nutrients will be estimated by Avanutri Revolution® Nutritional Evaluation software (Rio de Janeiro, Brazil) [27].
Physical activity: levels of physical activity will be evaluated according to the International Physical Activity Questionnaire (IPAQ) short version [28] translated and validated into the Portuguese language.
Laboratory variables
Blood samples will be collected by a trained professional at baseline and after 12 weeks. Samples will be centrifuged at 22 °C, 2000 rpm, for 10 min and stored in Eppendorf tubes at −80 °C for later analysis.
Lipid profile: total cholesterol, low-density lipoprotein cholesterol (LDL-c) and serum triglycerides will be evaluated by the enzymatic colorimetric method; high-density lipoprotein cholesterol (HDL-c) will be evaluated by the immunoprecipitation method (Roche Modular P Chemistry Analyzer®).
Inflammatory profile: interleukin-6 (IL-6), interleukin-10 (IL-10) and tumor necrosis factor alpha (TNF-α) will be evaluated by enzyme-linked immunosorbent assay (ELISA) using commercial kits (R&D Systems, Inc., Minneapolis, MN, USA), and according to the manufacturer’s instructions in a spectrophotometer (SpectraMax M2®). High-sensitivity C-reactive protein (hs-CRP) will be evaluated by the turbidimetric technique (Roche Cobas Integra 400 Plus Chemistry Analyzer®). Fibrinogen will be evaluated by the coagulometric method (Sysmex CA-600 systems®).
Glycemic profile: fasting glucose will be evaluated by the enzymatic colorimetric method (Roche Modular P Chemistry Analyzer®). Glycated hemoglobin will be evaluated by the immunoturbidimetric method (Roche Cobas Integra 400 Plus Chemistry Analyzer®). Serum insulin will be evaluated by electrochemiluminescence technique (Roche Elecsys 2010 Immunoassay Analyzer®).
Outcome measures
Primary outcome
The primary outcome measure will consist of changes in LDL-c (mg/dl) from baseline to 12 weeks.
Secondary outcomes
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Changes in other lipid measures (total cholesterol, HDL-c, serum triglycerides) and inflammatory profiles (hs-CRP, IL-6, IL-10 and TNF-α) from baseline to 12 weeks.
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Changes in glycemic profile (fasting glucose, serum insulin and glycated hemoglobin), anthropometric indexes (weight, BMI and waist and neck circumferences) and in systolic and diastolic blood pressure from baseline to 12 weeks.
Patients will be prompted to complete the follow-up; if this is not possible, a 12-week visit will be scheduled for blood collection and measurements of primary and secondary outcome variables.
Sample size
Sample size calculation was carried out in the WinPepi 11.20 for Windows program (Brixton Health, Israel). We used the data reported by Casas et al. [9], which assessed the effectiveness of a Mediterranean Diet pattern compared to a control diet among 164 individuals at high risk for cardiovascular disease. For a significance level of α = 0.05 and a statistical power of 80 %, with a between-group difference corresponding to a reduction of 13 mg/dl in the primary outcome (LDL-c) and considering no differences between olive oil and nut diets, the minimum sample size would be 171 patients (57 randomized to each group). Considering an expected loss rate of 20 % of patients, the total sample size needed will be 204 patients.
The allele frequency of the rs1761667 G>A and rs8069645 A>G SNPs in a Brazilian population is unknown. Since, in other studies, a frequency of approximately 40 % for each allele (A and G) has been found [23, 24], we expect that 204 individuals will provide a statistical power of 95 % in this analysis.
Statistical analyses
All data will be double-checked prior to statistical analyses. Baseline demographic and clinical characteristics of the participants according to groups will be analyzed using descriptive statistics, and inferential statistics will be employed to compare the effects of the dietary interventions on the different outcome measures. Continuous variables following a normal distribution will be expressed as mean ± standard deviation and asymmetrically distributed continuous variables will be expressed as median and interquartile range. Categorical variables will be expressed as absolute and relative frequencies. Departure of genotype distributions from the Hardy-Weinberg equilibrium in all participants will be assessed using chi-square tests. For between-group comparisons, analysis of variance (ANOVA) and the Kruskal-Wallis H test will be used for variables with normal and asymmetric distribution, respectively. The chi-square or Fisher’s exact tests will be used to evaluate categorical variables. All relevant endpoints (new cardiovascular events and death) will be registered as absolute number and percentages according to groups. The generalized estimating equations (GEE) model under the missing-at-random assumption will be used for comparison between outcome variables during the study period. Delta from all outcome data will be obtained from the difference between the measure at 12 weeks and baseline, and will be compared according to groups using analysis of covariance (ANCOVA), adjusted for potential confounders (for instance, differences in baseline data). The SNPs will be included in all ANCOVA models as interaction variables. There is no interim analysis planned. The significance level will be set at 5 %, and all data will be analyzed in SPSS 18.0 (SPSS Inc., Chicago, IL, USA) according to intention-to-treat.