Impact of Beef on Metabolites and Inflammation

The approach for this study is to enroll a population of healthy individuals (n=10; 5 men and 5 women) in a double-blind, two-armed (Organic Grass-fed and Conventional-fed), randomized, crossover trial in which each individual participant serves as their own control to compare postprandial serum bile acid responses between steak meals, one which is organically grass-fed (OGF) and the other conventionally-fed (CONVF). Participants will consume a 6-oz steak meal grilled to 158°F, the recommended temperature by the USDA. The randomized crossover design with half of the participants starting in the OGF arm and the other half starting in the CONVF arm will eliminate potential order effects. Block randomization with 2 blocks (total of 5 per block) will be created. Having each person serve as their own control in a crossover trial will eliminate the influence of inter-individual variation. The MSU Nutrition Research team has extensive experience with postprandial testing protocols and dietary manipulation and will utilize this experience to perform the proposed protocol with appropriate pre-test standardization, uniformity of meal preparation, precise timing of blood collection, and established procedures for processing and analyzing blood samples. Bile acids, including all primary and secondary forms, will be analyzed from samples collected before the meal and at 30-minute intervals for 4 hours after the meal. Established methods in the MSU Proteomics, Metabolomics, and Mass Spectrometry Facility will be used to compare total and individual bile acids between OGF and CONVF conditions.

Procedures:

During Visit 1, participants will undergo informed consent, be asked to complete a series of written questionnaires and anthropometric measurements. There will be a minimum 72 run-in period just prior to Visit 2, during which participants will be asked to follow certain dietary guidelines, such as no red meat. The second (Visit 2) and third (Visit 3) visits will take approximately 5 hours each and will each include a total of 9 blood draws through a sampling catheter, with each blood draw collecting about 2-3 teaspoons of blood. Blood will be drawn before a provided steak meal then every thirty minutes for 4 hours after meal consumption. There are a minimum of 72-hours between visits 1 and 2, and 7 days between visits 2 and 3. During this minimum 72- hour and 7-day period, participants will be asked to follow provided dietary and physical activity instructions. In addition, participants will be asked to complete a one month diet history questionnaire and a 24-hour dietary recall.

Dietary intervention: A meat science specialist will select and prepare the steaks which will be given to participants. Participants will be asked to consume a 6-ounce beef steak meal, cooked to 158°F that is either OGF or CONVF, but will be coded and labeled to ensure blinding.

Kinetics (measures over time) and untargeted and targeted metabolomics (an advance analytical technique to measure 1000's of molecules in a single sample) will be used as a tool to differentiate potential health impacts of the separate meal tests to investigate the influence of organic grass-fed beef versus conventionally-fed beef consumption on fasting and postprandial responses in healthy adults. The investigators expect to identify an array of metabolic differences between beef samples and will identify known impacts of those differences on health to provide a substantive comparison and provide a foundation for future research.

Metabolomic and Lipidomic analysis: When all samples have been collected, the serum samples will be thawed, and a metabolite fraction will be extracted. After extraction, the samples will be explored using mass spectrometry analysis. An initial targeted analysis will focus on bile acids, lipids, and amino acids. Targeted analytes will be quantified using standard curves and the resulting concentrations will be compared using several statistical analysis tools. Metabolite samples will be analyzed using a Waters Synapt G2Si QTOF (Waters Corporation). This instrument has ion mobility capability and can differentiate closely related compounds, such as bile acids, in complex mixtures based on their cross-sectional area and mass. The high sensitivity and variety of scan modes make the Synapt QTOF the optimal choice for untargeted analysis of complex biological samples.

Dietary analysis: This study will use the most recent version (2018) of the web-based Diet History Questionnaire (DHQ III), a food frequency questionnaire designed for adults 19 and older, developed by staff at the Risk Factor Monitoring and Methods Branch (RFMMB) of the NIH National Cancer Institute. The outputs of the DHQ III include carbohydrate constituents, carotenoids and tocopherols, dietary constituents from supplements, fats, fatty acids and cholesterol, macronutrients and energy, minerals, protein constituents, and vitamins are dietary constituents and food groups available in the DHQ III output files. In addition, acute dietary consumption will be determined using the free, web-based Automated Self-Administered 24-hour (ASA24®) questionnaire.