Performing Department
(N/A)
Non Technical Summary
The Dietary Guidelines for Americans recommendation to choose fat-free and low-fat in place of full-fat dairy products has become highly controversial, as has the potential mitigating effect of fermentation. Within this context the question has arisen as to whether the current guidance regarding dairy products for heart disease risk reduction should be modified. To clarify this issue a study was designed to compare the effects of full-fat and fat-free dairy, with and without fermentation (yogurt), on heart disease risk factors and determine if the effects observed are mediated by the bacteria in our intestine (gut microbiome) and the components they produce. To accomplish this we will conduct a randomized-controlled cross-over trial comparing the effect of consuming two servings/day of full-fat milk, fat-free milk, full-fat yogurt and fat-free yogurt on heart disease risk factors (e.g., blood lipid and glucose concentrations) and the gut microbiome composition and its products. Heart disease and related disorders are the leading cause of death and disability in the U.S. Poor diet quality is a major contributor. It is more critical that ever to have the evidence base on which to formulation the best advise.
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
0%
Goals / Objectives
Long-term goal: The longstanding Dietary Guidelines for Americans recommendation to choose fat-free/low-fat dairy in place of full-fat dairy has become highly controversial, as has the potential mitigating effect of fermentation. To address this controversy, our long term goal is to increase insights into cross-talk between host diet, gut microbiome, gut and serum metabolome and indicators of cardiometabolic health. The overarching goal of this proposal is to compare full-fat and non-fat dairy, with and without fermentation, on cardiometabolic risk factors and determine if differences are mediated by functional alterations in gut microbiome and derived metabolites. To accomplish this goal we propose to conduct a randomized-controlled cross-over trial to compare the effect of consuming two servings per day of full-fat (bovine) dairy (milk), fat-free dairy (milk), full-fat fermented dairy (yogurt), and fat-free fermented dairy (yogurt) to address the following objectives.Objective 1. Determine effect of dairy fat in milk, with and without fermentation, on fecal microbiome composition, function and diversity, and measures of gut barrier integrity.Hypothesis 1: Unfermented full-fat, compared to unfermented fat-free milk, will result in (i) lower microbial diversity, (ii) higher abundance of bile salt hydrolase (e.g., Lactobacillus, Bifidobacterium, Enterococcus, Clostridium spp. and Bacteroides) and lipophilic (e.g. Eubacterium coprostanoligenes, Bacteroides intestinalis and Faecalibacterium prausnitzii) microbiota, (iii) lower abundance of butyrate producing microbiota (e.g., Faecalibacterium prausnitzii and Clostridium leptum of the family Ruminococcaceae, and Eubacterium rectale, Roseburia spp. of the family Lachnospiraceae, Eubacterium hallii and Anaerostipes spp) and proteolytic microbiota (e.g., Lactococcus lactis, Lactobacillus, Leuconostoc, Pediococcus, and Streptococcus gener), and (iv) lower gut barrier integrity (higher lipopolysaccharide [LPS] and lipopolysaccharide binding protein [LBP] concentrations). These alterations will be favorably modulated by fermentation.Objective 2. Determine the effects of dairy fat in milk, with and without fermentation, on fecal and serum metabolomes, and CMRF.Hypothesis 2: Full-fat, compared to fat-free, unfermented milk will (i) increase fecal trimethylamine (TMA) and (serum trimethylamine oxide [TMAO]), 1° and 2° bile acids, com-plex lipids (cholesteryl ester, phosphatidylcholine, lysophosphatidylcholine, ceramides, sphingomyelins, diglycerides/triglycerides]), and decrease branched chain amino acids [BCAA] and short chain fatty acids [SCFA]), and (ii) these responses will be associated with higher fasting serum insulin, low density lipoprotein (LDL)-cholesterol and triglyceride concentrations, Homeostatic Model Assessment for Insulin Resistance (HOMA-IR), systolic and diastolic blood pressures, and inflammatory biomarkers (high sensitivity C-reactive protein [hsCRP] and tumor necrosis factor alpha [TNFα]) concentrations. These alterations will be favorably modulated by fermentation.Objective 3. Determine associations between gut microbiome with the fecal metabolome, serum metabolome and CMRF using bioinformatics/integrative pathway analysis to identify biological pathways modulated by dairy fat and fermentation.Hypothesis 3: The differential effects of full-fat and fat-free fermented and unfermented dairy on CMRF risk factors will be mediated by gut microbiota and derived metabolites on host glucose, amino acid, lipid and inflammatory metabolic pathways.Exploratory Objective. Assess the effect of sex as a biological variable on responses to the dietary interventions.Exploratory Hypothesis: Women will have higher microbial composition, diversity and functional rich-ness than men.
Project Methods
Study protocol: Two servings per day of the intervention dairy items (hence forth referred to as items) will be provided to study participants according to a randomized-controlled cross-over design. Block allocation will be used to assign randomization sequence. Body weight and blood pressure will be measured at each visit to maximize participant/staff interaction. Each intervention phase will be 3 weeks, with a 2 week wash-out period between phases.Participant characteristics: To focus on a clinically relevant population yet with-in the context of maximizing generalizability the target cohort (N=20, 50%/50% F/M) will have the following characteristics; >50 years (women postmenopausal), BMI >25-35, no chronic antibiotics use, no milk allergies or lactose intolerance, not vegan, no chronic gastrointestinal disorders, and not taking lipid lowering medications or dietary supplements (other than multivitamins). Study participants must voluntarily agree to follow the protocol as described and approved by the Tufts University/Tufts Medical Center Institutional Review Board (IRB), and sign an IRB approved consent form.Sample collection: Fecal samples will be collected at the start and week 3 of each diet intervention phase. Once produced, samples will be brought to the MRU (vouchers provided). Samples will be processed, aliquoted and stored at -80°C for metagenomic, SCFA and TMA analyses. A portion will be freeze-dried, aliquoted and archived for metabolomic analysis. Fasting (12 hour) and post-prandial (4 hour) blood samples will be collected at end of each 3-week intervention phase. Serum and plasma will be isolated and stored at -80°C for metabolome and CMRF analyses, respectively.Dietary intake recalls: Two 24-hour diet recalls will be conducted during each intervention phase and analyzed using Nutrition Data System for Research (NDSR software versions 2022 and 2023). These data will be used to assess intervention compliance, food displacement, and estimate energy and nutrient intakes.Gut microbiome taxonomic profiling (diversity and abundance) and functional pathway analysis using shotgun metagenomic sequencing of fecal samples, and gut barrier integrity.Gut microbiome: Gut microbiome composition and function data will be generated by CosmosID Inc. Specifically, CosmosID will perform DNA extraction from 160 stool samples, sample quality control, standardized library preparation and 12 M read metagenomics sequencing. Fecal DNA will be extracted (QIAGEN DNeasy PowerSoil Pro), then quantified using fluorometer Qubit 4. Each DNA sample will be normalized and libraries constructed using their established Library Prep kit. The libraries will be pooled by adding an equimolar ratio of each based on bioBakery.Gut barrier integrity (LPS, LBP): Serum LPS concentrations will be measured using a homogenous enzymatic assay (EndoZyme Recombinant Factor C Endotoxin Detection Assay, Hyglos GmbH of bioMérieux, Bernried, Germany) and LBP concentrations using an enzyme-linked immunosorbent assay kit (Hycult Biotech Inc, Wayne, PA).Fecal and serum metabolome - targeted metabolomicsFecal metabolome: A semi-targeted approach using liquid chromatography-quadrupole time-of-flight/mass spectrometry (QTOF) will be used to screen and quantify the fecal metabolome. Metabolites will be extracted from freeze dried fecal samples using ethanol/ phosphate buffer and loaded on a 96-well sample preparation plate (Biocrates MxP Quant 500). After derivatization using phenyl isothiocyanate, target analytes will be extracted and diluted inside the wells following the manufacturer's protocol, and measured by flow injection analysis (FIA) on an Agilent 6550 QTOF high resolution mass spectrometer with an electrospray ionization source. Data will be quantified using Agilent MassHunter Workstation Quantitative Analysis for QTOF Version 10.1. Metabolites will be reported as absolute concentrations using pre-loaded sets of defined standards as well as judiciously selected fecal metabolites based on the available literature and databases (e.g., KEGG, Human Metabolome Project, LIPID MAPS, PubChem, CAS).Serum metabolome: Using the same semi-targeted QTOF methodology and Biocrates kit described for the fecal metabolome, the serum metabolome will be analyzed for the following metabolites: amino acids and related metabolites; biogenic amines; bile acids; acylcarnitines and complex lipids (phosphatidylcholines, lysophosphatidylcholine, sphingomyelins, ceramides, cholesteryl esters, diglycerides and triglycerides). Absolute concentrations will be derived using internal standards, quality control samples and external calibrations standards.Short chain fatty acids: Acetate, propionate, butyrate, isobutyrate, 2-methyl butyrate, valerate, isovalerate, 3-methylvalerate, 4-methylvalerate) will be quantified in serum and fresh fecal samples after derivatization with 3-nitrophenylhydrazine. They will be measured using a QTRAP 5500 LC-MS/MS method .TMA and TMAO: TMA in fecal, and TMA and TMAO in plasma samples, will be determined by a QTRAP 5500 LC-MS/MS method .Cardiometabolic risk factors: Plasma fasting and 4-hour post-prandial total cholesterol, HDL-cholesterol, triglyceride and glucose concentrations will be measured using an automated clinical chemistry analyzer (AU480, Beckman Coulter Inc. CA). LDL-cholesterol concentrations will be calculated using the Friedewald formula, except when triglyceride concentrations exceed 400 mg/dL, in which case a direct LDL-cholesterol two reagent colorimetric enzymatic assay will be used (AU480, Beckman Coulter, Inc., CA). Serum insulin and hsCRP concentrations will be measured using solid-phase, two-site chemiluminescent immunometric assays (IMMULITE 2000, Siemens Healthcare Diagnostics, Los Angeles, CA).Plasma dairy fat biomarkers: Plasma phospholipid fatty acid profiles will be determined using a previously established gas chromatograph methodology. Peaks of interest will be identified by comparison with authentic fatty acid standards (Nu-Check-Prep) and expressed as molar percentage (mol %) and proportions relative to internal standards.