Impact of Cottonseed Oil on Oxidative Stress, Inflammation, and Metabolism
Condition: Inflammation; Healthy; Oxidative Stress; Metabolic Syndrome
MeSH terms: Metabolic Syndrome; Inflammation
Study type: Interventional

Summary: 
      Recent research evidence suggests that cottonseed oil (CSO) may have both direct and indirect
      anti-inflammatory and anti-oxidative impacts linked to bioactive components of CSO and
      favorable alterations in lipid metabolism. These impacts are directly related to
      non-communicable diseases such as diabetes, cardiovascular diseases, and cancer. Our
      overarching hypothesis is that the effect of CSO consumption on oxidative stress markers
      (isoprostanes), inflammatory cytokines, metabolic biomarkers, and bile acid metabolism will
      be beneficial for reversing disease pathophysiology linked to oxidative stress, inflammation,
      and bile acids. Our long-term goal is to establish effective and practical therapeutic
      strategies utilizing dietary incorporation of CSO to prevent or reverse these diseases.

      The following hypotheses will be tested in the proposed investigation:

      H1: CSO consumption will lower exercise-induced oxidative stress, and the effect of CSO will
      be greater than that of OO for lowering of exercise-induced oxidative stress.

      H2: CSO consumption will lower inflammatory cytokines and metabolic markers linked to the
      inflammation process in human participants, and the effect of CSO will be greater than that
      of OO for lowering inflammation.

      H3: Features of serum bile acids, serum metabolomes, and lipidomes distinguishing CSO and OO
      treatment correspond to metabolic pathways illuminating the health benefits of CSO treatment.

      H4: Metabolic and inflammatory impacts of dietary oils will be greater for 60 g/d of CSO
      compared to 30 g/d.
    

Detailed description: 
      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.

      Metabolomic 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 and a
      set of inflammation and oxidative stress biomarkers including 8-isoPGF2a. Targeted analytes
      will be quantified using standard curves and the resulting concentrations will be compared
      using several statistical analysis tools. A deep untargeted analysis will also be undertaken
      which may reveal additional metabolic pathways modulated by CSO consumption. 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: Long-term dietary habits may create adaptations that influence the response
      to the short-term supplementation of CSO and OO. 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.

      Statistical test: H1 will be tested using analysis of variance (ANOVA) to compare the
      differences in exercise-induced oxidative stress markers (isoprostanes) among the individuals
      consuming either 30 g CSO, 30g OO, or 60 g CSO, or 60 g OO. H2 will be tested using analysis
      of variance (ANOVA) to compare the differences in inflammation and inflammatory markers among
      the individuals consuming either 30 g CSO, 30g OO, 60 g CSO, or 60 g OO. To test H3, the
      investigator will identify changes in serum bile acids and metabolomes and then determine the
      metabolic pathways associated with the metabolic changes to identify potential mechanisms
      underlying the health impacts of CSO. To test H4, the investigator will identify the measure
      of serum isoprostanes, bile acids, inflammation, lipidomes, and metabolomes, and then use
      regression analysis to determine the level of variability in response to the different doses
      of CSO and OO.
    

Intervention name(s): High dose CSO; Low dose CSO; Low dose OO; High dose OO
Intervention type(s): Behavioral; Behavioral; Behavioral; Behavioral
Intervention description(s): Participants in the high-dose CSO arm of the study will consume two smoothies containing a total of 60 g of CSO per day for 4 weeks.; Participants in the low-dose CSO arm of the study will consume two smoothies containing a total of 30 g of CSO per day for 4 weeks.; Participants in the low-dose OO arm of the study will consume two smoothies containing a total of 30 g of OO per day for 4 weeks.; Participants in the high-dose OO arm of the study will consume two smoothies containing a total of 60 g of OO per day for 4 weeks.
Other intervention name(s): HD CSO; LD CSO; LD OO; HD OO
Allocation: Randomized
Intervention model: Parallel Assignment
Intervention model description: The design is randomized parallel design, where the healthy adults will be randomized into 30 g CSO, 30 g OO, 60 g CSO, and 60 g OO.
Masking: Double (Participant, Investigator)
Primary purpose: Prevention