The Dose-response Effect of a Four-week Intervention of Low and High Dose of Cottonseed Oil on Exercise-induced Oxidative Stress, Inflammation, and Metabolism in Healthy Adults

The approach will be to enroll a population of healthy individuals (n=32) in a four-armed (30 g CSO, 30 g OO, 60 g CSO, and 60 g OO), randomized, double-blind, parallel controlled trial to compare the effect of these different doses of oils on exercise-induced oxidative stress.

During Visit 1, participants will undergo informed consent, anthropometric measurements, and a sub-maximal exercise test to determine their VO2max. Participants will also fill Global physical activity questionnaire (GPAQ), exercise readiness questionnaire (2022-PARQ+), AHA/ACSM Health/Fitness Facility Pre-participation Screening Questionnaire, and menstruation-related questionnaire. There will be a minimum of three days run-in periods between Visit 1 and Visit 2, during which participants will be asked to follow certain dietary guidelines such as minimal use of oil, limiting the fatty fish, omega-3, vitamin A, vitamin C, vitamin A supplements, and limiting the consumption of nuts/nut seeds/oils/oilseeds or foods containing these components. During Visit 2, participants will do an exercise test (75% VO2max for 30 mins), and a blood draw will be done before and after the exercise test. Blood will be drawn after 15 minutes of the exercise test. In addition, participants will be asked to do one month diet history questionnaire and 24 hours of dietary recall. There will be four weeks between Visit 2 and Visit 3, during which period participants will be asked to consume smoothies containing respective doses of oils every day. We will provide smoothies every week, and an additional oil will be provided in an aluminum-foil-wrapped squirt bottle for participants to substitute with their regular oil. In addition, participants will be asked to fill out a compliance check form to confirm their compliance with the study diet. There will be one weekly question on gastrointestinal issues in the compliance form. After four weeks of dietary intervention, participants will come to the lab for Visit 3. Visit 3 will follow a similar protocol to Visit 2 except that the anthropometric measurements will be done in Visit 3 as well.

Procedures:

Exercise test: During Visit 1, a sub-maximal exercise test will be done to predict how much oxygen the participant's body is able to use during exercise. This allows the researchers to have one participant exercise at the same submaximal percent of his/her maximum as all of the other participants, based on his/her fitness level. This will begin with walking on a treadmill (Woodway GmbH D79576, Weil am Rhein, Denmark) for 5 minutes at a self-selected comfortable speed at 0% grade to warm up before the test. The test involves a series of stages that get increasingly more difficult every two minutes. Participants will wear a heart rate monitor, breathe through a mouthpiece that is connected to an analyzer, and wear a nose clip. The test begins with walking, and as the participant move to more difficult stages, the intensity is increased by increasing grade and speed. The researcher will end the test when the participant has reached 85% of their age-predicted maximal heart rate (220-age in years). During visits 2 and 3, participants will report to the laboratory after an overnight fast, and blood samples will be collected before and after an exercise test. The exercise test will involve a brisk uphill walk at 75% VO2max for 30 minutes on a treadmill. During exercise, a metabolic measurement system (ParvoMedics TrueMax 2400 Metabolic System, Sandy, Utah, USA) will be used to determine a respiratory quotient and the associated level of fat oxidation. The respiratory quotient will be measured by determining the volume of carbon dioxide released over the volume of oxygen absorbed during respiration. After the blood collection, TG (and full lipid panel plus glucose) will be analyzed in real-time using a clinical chemistry analyzer (Piccolo press). At the same time, serum samples will be aliquoted and stored at -80 C until analysis for inflammatory cytokines (a profile of established mediators and biomarkers), insulin, other metabolic biomarkers linked to inflammation, and the lipidomics and metabolomics analysis. For the analysis of isoprostanes, serum samples will be snap-frozen in dry ice and ethanol slurry after it is aliquoted and immediately stored at -80 C. Investigators will measure inflammatory cytokines (TNF-α, interleukin (IL)-1β, IL-6, IL-7, IL-23, and granulocyte-macrophage colony-stimulating factor (GM-CF) using high-sensitivity Luminex multiplexing technology (Bio-Rad Bio-Plex 200 HTS) prepared by Millipore.

Dietary intervention: A registered dietitian will prepare a study diet (smoothies) which will be given to participants for four weeks. Participants will be given smoothies containing either 30 g CSO, 30 g OO, 60 g CSO, or 60 g OO per day in two separate containers. The 30 g smoothies (both CSO and OO) contain 762 kcal Calories, 120 g Carbohydrates, 3 g Protein, and 30 g Fat. The 60 g smoothies (both CSO and OO) contain 761 kcal Calories, 50 g Carbohydrates, 3 g Protein, and 30 g Fat. During these four weeks period, participants will be asked to follow certain dietary guidelines and will be asked to fill out a compliance form. Participants will be asked to fill out the compliance check form daily, indicating how much smoothie they consumed and if they consumed anything not recommended by the research team. Smoothies will be given weekly, meaning participants will visit the lab every seven days to pick up their smoothies and drop off the weekly compliance form.