Recipient Organization
COLORADO STATE UNIVERSITY
(N/A)
FORT COLLINS,CO 80523
Performing Department
Food Science & Human Nutrition
Non Technical Summary
Aging is the primary risk factor for cardiovascular disease largely due to adverse changes to the arteries. Aside from aging, numerous factors increase cardiovascular disease risk including diet and nutrition. Specifically, consumption of a high-fat diet is a contributing factor. Adverse responses to a high-fat meal may include arterial dysfunction, and increases in blood triglyceride levels, glucose, inflammation and free radicals. These responses are exacerbated in individuals who already suffer from excessive body weight and other cardiovascular disease risk factors such as hyperlipidemia and hyperglycemia. This can have significant adverse effects on cardiovascular health. As such, interventions that can prevent or reduce these responses to a high-fat diet in general, or the consumption of a high-fat meal, can play an important role in preserving cardiovascular health and reducing cardiovascular disease risk. Red beetroot juice is an excellent source of nutrients and phytochemicals. These compounds have been shown to have free radical quenching, anti-inflammatory, and cardiovascular-protective effects. Given the findings of previously conducted research in the broad area of red beetroot consumption and human health, it can be suggested that: 1) acute red beetroot consumption may prevent or reduce the disturbed responses to consuming a high-fat meal in individuals with exaggerated responses; and 2) chronic consumption of red beetroot may improve underlying factors contributing to these exaggerated responses (e.g. improvements in vascular health). It is likely that the compounds in red beetroot juice work synergistically than indepdently. Accordingly, the overall goal of the proposed project is to investigate the efficacy of acute and chronic whole red beetroot juice consumption in reducing adverse responses to the consumption a high-fat meal and to gain insight into the reasons responsible for its effects.This work is important because the findings will inform the public on an area with important nutrition, food, and human health relevance. As such, individuals can use this information to make well-informed decisions about their diets and the inclusion of certain foods (e.g. functional foods) that can improve their nutritional status and thus their overall health and well-being.
Animal Health Component
45%
Research Effort Categories
Basic
45%
Applied
45%
Developmental
10%
Goals / Objectives
Aging is the primary risk factor for CVD largely due to endothelial dysfunction which is involved in the development and progression of a number of cardiovascular disorders including atherosclerosis, hypertension, and arterial stiffness. Aside from aging, numerous factors increase CVD risk including diet and nutrition. Specifically, consumption of an atherogenic (e.g. high-fat) diet is a contributing factor. Postprandial cardiometabolic responses to a high-fat meal may include endothelial dysfunction (characterized by impaired endothelial-dependent vasodilation secondary to reduced nitric oxide [NO] bioavailability), dyslipidemia, hyperglycemia, inflammation and oxidative stress. These responses are exacerbated in individuals who already suffer from excessive body weight and other CVD risk factors such as hyperlipidemia and hyperglycemia. Considering that much of the day is spent in the postprandial state, this can have significant detrimental effects on cardiovascular health. As such, interventions that can prevent or mitigate these responses to a high-fat diet in general, or the consumption of a high-fat meal, can play an important role in preserving cardiovascular health and reducing CVD risk. Red beetroot (Beta vulgaris L.) is an excellent source of bioactive compounds including nitrate, flavonoids, phenolic acids, betalains, carotenoids, and ascorbic acid. These bioactive compounds have been shown to have antioxidative, anti-inflammatory, and cardiovascular-protective effects. Given the findings of previously conducted research in the broad area of red beetroot consumption and human health, it can be suggested that: 1) acute red beetroot consumption may prevent or attenuate the disturbed postprandial responses to consuming a high-fat meal in individuals with exaggerated responses; and 2) chronic consumption of red beetroot may improve underlying factors contributing to these exaggerated responses (e.g. chronic endothelial dysfunction). Because dietary nitrate is reduced to NO through the enterosalivary nitrate-nitrite-NO pathway independently of the vascular endothelium, much credit has been given to its nitrate content rather than the other bioactive compounds which likely work synergistically. Accordingly, the overall goal of the proposed project is to investigate the efficacy of acute and chronic whole red beetroot juice consumption in attenuating adverse postprandial responses to a high-fat meal and to gain insight into the underlying mechanisms responsible. Our central hypothesis is that acute and chronic consumption of whole beetroot juice with all of its bioactive components will be more effective than potassium nitrate, nitrate-free beetroot juice, and placebo in attenuating the negative effects of a high-fat, mixed macronutrient meal on endothelial and cardiometabolic function. This hypothesis will be tested using the following specific aims:Specific Aim 1: To determine the extent to which whole beetroot juice will attenuate acute endothelial dysfunction following consumption of a high-fat, mixed macronutrient meal. This aim will be assessed by measuring reactive hyperemia index using peripheral arterial tonometry.Specific Aim 2: To investigate the extent to which whole beetroot juice will prevent cardiometabolic disturbances following consumption of a high-fat, mixed macronutrient meal. This aim will be assessed by measuring postprandial brachial blood pressure, lipemia (blood triglyceride [TG] concentrations), glycemia (blood glucose and insulin concentrations). Specific Aim 3: To identify potential mechanisms by which whole beetroot juice exerts its effects on vascular endothelial function and cardiometabolic health. This aim will be assessed by measuring oral nitrate-reducing bacteria, plasma and salivary NO metabolites, plasma and urinary polyphenol metabolites, and blood biomarkers of oxidative stress (oxLDL) and inflammation (interleukin-6 [IL-6] and lipopolysaccharide [LPS]).
Project Methods
Study Overview: We propose a randomized, double-blind, placebo-controlled, 4-period, 4-week crossover pilot clinical trial consisting of 2 postprandial tests for each period. 15 overweight or obese postmenopausal women and men aged 40 to 65 will be recruited from the greater Fort Collins, CO area. After telephone prescreening, participants will report to the study site for their first visit (Screening) where they will receive verbal and written explanation of the project, provide informed consent, followed by assessments of medical history, dietary intake, and physical activity. Qualified participants will be scheduled for a baseline visit and randomly assigned to their respective treatments. On the second visit (Baseline) following an overnight fast, anthropometrics and blood pressure will be measured and diet and physical activity records will be collected. Subjects will ingest their respective treatment 10 min prior to consuming the test meal. Endothelial function will be assessed at baseline (0 minutes) and 4 hours postprandially. Blood samples will be collected at baseline (0 minutes) and 1, 2, 4, and 6 hours postprandially. Urinary polyphenol metabolites will be assessed at baseline and 24-hours post intervention. Oral bacteria will be assessed at baseline and 4 weeks. At the third visit (Final), all assessments will be repeated at the same time points but 24 hours after consuming the last dose of their respective treatments to test chronic rather than acute effects. Subjects will undergo a 4-week washout period before crossing over to the next treatment period. This will be repeated for all 4 treatments.Subjects: Postmenopausal women and men aged 40 to 65 years and overweight/obese (BMI 25-40 kg/m2) will be included. Individuals taking antihypertensive, lipid-lowering, or hormone replacement medications, diagnosed hypertension, CVD, diabetes, cancer, kidney, liver, or pancreatic disease, in a weight loss program, heavy smokers (≥ 20 cigarettes/d) and drinkers (> 12 drinks/wk), allergy to meals/treatments, or taking > 2 servings beetroot juice/wk will be excluded.Medical History and Blood Pressure: Information on conditions, diseases, menstrual history, medication, hormone, and nutritional supplement use will be obtained. Blood pressure will be measured in triplicate using an automatic device (Omron Healthcare).Anthropometrics, Diet, and Physical Activity Assessment: Height, weight, BMI, waist and hip circumferences will be measured using NHANES III methods. Three-day food records will be analyzed using NutritionistProTM diet analysis software (Axxya Systems). Physical activity patterns will be assessed using a seven-day physical activity recall.Blood, Urine, and Saliva Collection and Processing: Venous blood will be collected in vacutainers with appropriate anticoagulants, centrifuged, aliquoted, and kept at -80°C. Fasting midstream urine will be collected in sterile cups, aliquoted and kept at -80°C. Saliva will be collected, centrifuged, and supernatant kept at -80°C. Buccal swabs will be kept at -80°C.Test Meal: The test meal will contain 900 kcal and the macronutrient composition will be 50% fat (450 kcal, 50 g), 32% kcal from carbohydrates (288 kcal, 72 g), and 18% protein (162 kcal, 41 g). It will consist of a bagel, butter, cream cheese, jelly, boiled egg, and whole milk. Treatments and Compliance: Treatments: 1) 70 mL isocaloric placebo (PBO), 2) 70 mL PBO + 400 mg potassium nitrate (NIT); 70 mL beetroot juice concentrate (BRJ; contains 400 mg NIT), or 3) 70 mL nitrate-free BRJ (NF-BRF). All treatments will be provided by James White Drinks, Ltd. The PBO will be made isocaloric to the BRJ with similar degrees of sweetness, flavor and color. Compliance will be monitored examining unused treatments and a daily treatment log.Endothelial Function: Small finger artery endothelial-dependent vasodilation will be assessed using a non-invasive plethysmographic method (EndoPAT2000, Itamar Medical, Ltd) at baseline (0 minutes) and 240 min postprandially. Maximal changes in endothelial function occur 3-4 hours after a high-fat meal. After 10 min rest, the blood pressure cuff on the study arm will be inflated to 60 mmHg above systolic blood pressure for 5 min. The cuff will then be deflated in to induce reactive hyperemia and the signal from both PAT channels will be recorded by a computer. The reactive hyperemia index (RHI), an index of flow-mediated vasodilation, will be derived as the ratio of the average pulse wave amplitude during hyperemia (60 to 120 sec of post-occlusion period) to the average pulse wave amplitude during baseline in the occluded hand, divided by the same value in the control hand and then multiplied by a baseline correction factor. A RHI of 1.67 indicates endothelial dysfunction. Framingham RHI (F-RHI) will be calculated using a different post-occlusion hyperemia period (90 to 120 sec) without the baseline correction factor. Diastolic augmentation index (dAix) will be generated using the shape of the pulse wave recorded by the probes during baseline and normalized for a 75 bpm heart rate of (dAix@75).Biomarkers: Glucose (YSI 2300 Stat Plus™ Glucose Analyzer, Yellow Springs Instruments), insulin (chemiluminescent immunoassay, Beckman Access 2 Analyzer, Beckman Coulter), TG (enzymatic assay, Beckman AU480 Chemistry Analyzer, (Beckman Coulter), NO (colorimetry, Cayman Chemical), IL-6, and oxLDL will be measured (ELISA, R&D Systems, and Mercodia) at all 5 time points. LPS will be measured at 0, 60 and 120 min (Kinetic-QCL assay, Lonza).Oral Bacteria: Nitrate-reducing oral bacteria will be quantified from buccal swabs by qPCR using species-specific primers. Gene copy numbers will be quantified by comparing to standard curves of amplified DNA from pure cultures obtained from the American Type Culture Collection.Metabolomics: Targeted detection and quantification of flavonoids, phenolics, and carotenoids will be conducted using UPLC-MS. Briefly, carotenoids will be extracted from plasma and urine using hexane, and then dried under N2, resuspended in mobile phase, and separated by isocratic reverse phase chromatography. Flavonoids and phenolics will be extracted using 9% saline and separated by using a binary mobile phase gradient reverse phase chromatography. All compounds will be quantified by comparing to standard curves of commercial compounds.Statistical Analyses: Given the pilot nature of this study, a power calculation to determine sample size is difficult and instead have based the sample size of 15 participants on previous similarly designed intervention studies. Statistical analyses will be performed using SAS V9.4 (SAS Institute). Baseline values will be compared using paired sample t-tests. The effects of treatments on outcomes parameters will be evaluated using repeated measures ANOVA. Mean changes during the postprandial periods will be examined by interaction effects of condition and time. Post-hoc comparisons will be made as appropriate using Tukey's tests. Statistical significance will be based p < 0.05 using two-tailed tests. Correlations between variables will be evaluated.