Flavonoids, Exercise, Gut-Derived Phenolics
Condition: Exercise and Gut-derived Phenolics
Study type: Interventional

Summary: 
      PURPOSE:

        1. PRIMARY: Using a randomized, parallel group design, this investigation will determine if
           the combination of 2-weeks polyphenol supplementation (using the Reoxcyn flavonoid
           supplement in capsule form) and one acute 45-minute brisk walking bout enhances the
           translocation of gut-derived phenolics into the circulation. A comparator group of
           runners (N=20) ingesting the flavonoid supplement for two weeks will be included (2.5 h
           run, 70% VO2max).

        2. SECONDARY: Data from this study will determine if 2-weeks supplementation of the Reoxcyn
           flavonoid supplement is a sufficient time period to increase circulating levels of
           gut-derived phenolics, extending data collected from the previous 12-week community
           trial.

      HYPOTHESIS: We hypothesize that an acute 45-minute brisk walking exercise bout (60% VO2max)
      following 2 weeks of increased polyphenol intake will be a sufficient exercise stimulus to
      increase gut permeability and the movement of gut-derived phenolics into circulation. If this
      hypothesis is confirmed, these data would have broad public health appeal, demonstrating that
      the combination of brisk walking and increased polyphenol intake augments circulating levels
      of health-restoring small phenolic molecules derived from bacterial breakdown in the colon.
    

Detailed description: 
      Research Design: Study participants (N=85) will be entered into the study and randomized to
      the flavonoid or placebo supplement groups for 2 weeks, with these two independent groups run
      in parallel (with N=80 anticipated to successfully complete all study phases). Supplements
      will be administered in a double-blinded manner in capsule form. Study participants will
      report to the research facility for baseline testing and orientation, and then pre-study and
      after 2-weeks supplementation, and then again 24 hour later. Four blood samples will be
      collected as shown in the Figure (pre- and post-supplementation, post-exercise/rest, and
      24-hour post-exercise/rest). When reporting to the lab after 2-weeks supplementation, study
      participants will be randomized to either sit or walk briskly on treadmills for 45 minutes. A
      comparator group of 20 runners ingesting the flavonoid supplement and running 2.5 h in the
      lab will be included to allow a comparison between moderate and intense exertion (and to
      provide pilot data for a future study). Baseline Testing: One to two weeks before the start
      of the study, study participants (walkers and runner) will be given an orientation to the
      study and then provide voluntary consent. Instructions will be given for recording all food
      and beverage intake in 3-day food logs (Thursday, Friday, and Saturday prior to starting
      supplementation). Demographic and training histories will be acquired with questionnaires.
      Height, body weight, and percent body fat (seca Medical Body Composition Analyzer 514
      bioelectrical impedance scale, Hanover, MD) will be measured. VO2max will be assessed using
      the Bruce's treadmill protocol, with oxygen consumption and ventilation continuously
      monitored using the Cosmed CPET metabolic system.

      Pre-Supplementation Lab Visit: On the first day of the study, participants (walkers and
      runners) will return to the lab in a fasted state (9 or more hours with no food or beverage
      other than water). Blood samples will be taken from an antecubital vein with subjects in the
      seated position. Participants (walkers) will be given a 2-week supply of flavonoid or placebo
      capsules organized into supplement trays to facilitate compliance. Runners will be given a
      2-week supply of flavonoid capsules. The 3-day food record will be reviewed and collected by
      the research team, and analyzed for nutrient and flavonoid content using the Food Processor
      v. 11.1 (ESHA Research, Salem, OR). Each food/beverage will be assessed for macro- and
      micro-nutrients, total flavonoids and subtotals for each of the six flavonoid subclasses, and
      two individual flavonoid values (quercetin, EGCG). During the 2-day period prior to visit #3
      to the lab, and during the entire day that includes visit #3, participants will be asked to
      avoid food and beverages containing sucralose (Splenda) and mannitol. To avoid sucralose and
      mannitol, participants will be asked to check ingredient lists for diet sodas, yogurt and
      other dairy products, snack foods, chewing gum and candy, low-sugar products, toothpaste, and
      mouthwash.

      Post-Supplementation Lab Visits:

      After the 2-week supplementation period, participants (walkers and runners) will return to
      the lab in an overnight fasted state (on the same day of the week as the pre-supplementation
      lab visit).

      Participants will turn in the supplement tray to verify compliance with ingesting the
      capsules. A blood sample will be collected. Participants (walkers) will be randomized to
      either sit for 45 minutes in the lab or to walk briskly for 45 minutes on a 5% graded
      treadmill at 60% VO2max (with metabolic monitoring during the first 5 minutes, and then at
      15, 30, and 45 minutes) using the Cosmed CPET metabolic cart. Runners will run at 70% VO2max
      on an ungraded treadmill for 2.5 h, with metabolic measurements made every 30 minutes. Water
      will be given ad libitum for all participants, with no other beverage or food allowed. Just
      prior to the sitting or walking lab sessions, and the 2.5 h run, participants will consume a
      150-ml solution containing 1 g sucrose, 1 g lactulose, 0.5 g L-rhamnose, 1 g erythritol, 1 g
      sucralose, 0.5 g mannitol.

      Urine will be collected in plastic containers for 24 h in two separate fractions: 0-5 h and
      5-24 h. No eating or drinking (except tap water) will be allowed during the first 5-h urine
      collection. Participants will return the next morning in an overnight fasted state, provide a
      blood sample, and turn in urine samples.

      Urine collection guidelines to the subjects:

        -  You should collect every drop of urine during each collection period (5 hours from the
           start of the exercise bout, and then the second collection lasting to the next morning
           or 5 to 24 h from the start of exercise). It does not matter how much or little urine is
           passed each time, as long as every drop is collected.

        -  Collect every drop of urine during the day and night in the two collection bottles (0-5
           hours, 5-24 hours, beginning with the start of the lab exercise test session). Store the
           bottles in the refrigerator. Be sure to collect any urine passed during bowel movements.

        -  Finish by collecting the first urine passed the next morning, adding it to the
           collection bottle. Bring both urine collection bottles with you to the lab (i.e., the
           morning after the exercise session when you will provide your final blood sample).

      Flavonoid Supplement: Supplement and placebo capsules will be prepared by Reoxcyn Innovation
      Group LLC (Salt Lake City, UT). (SEE supplementation information in attached file).
      Ingredients will be the same as tested in the community trial: vitamin C (as ascorbyl
      palmitate) (Green Wave Ingredients, La Mirada, CA), wild bilberry fruit extract (25% total
      anthocyanins) (FutureCeuticals, Momence, IL), green tea leaf extract (50% EGCG) (Watson
      Industries, Inc., Pomona, CA), quercetin aglycone (Novel Ingredients, East Hanover, NJ),
      caffeine (Creative Compounds, Scott City, MO), and omega 3 fatty acids (Novotech
      Nutraceuticals, Ventura, CA). Capsule fill ingredients and excipients include Nu-Flow 70R
      (from rice hulls), tapioca from cassava root, natural bamboo silica, marshmallow root.
      Placebo capsules will contain only the fill ingredients and excipients (without the active
      ingredients). The daily serving for the walkers will be 2 flavonoid or 2 placebo capsules.
      Two flavonoid capsules provides 329 mg flavonoids (American adult average, depending on the
      study, is 240 mg per day). The daily serving for the runners will be 4 flavonoid capsules
      (658 mg flavonoids). Participants will be given a 2-week supply of the either the flavonoid
      or placebo capsules, with instructions on how to consume daily in split doses (1 with
      breakfast and 1 with lunch, or double that amount for the runners). Study participants will
      be allowed to add one extra capsule per day to increase consumption if one or two days of
      taking the supplement are missed. Study participants will ingest 2 capsules (4 for the
      runners) just after providing the blood sample during the walk/rest (or run) lab session.

      SUPPLEMENT CAPSULES will be TESTED for flavonoid content pre- and post-study by Dr. Mary Ann
      Lila (NCSU, Plants for Human Health Institute, NCRC).

      Targeted Metabolomics Analysis:

      Gut-derived phenolic metabolites will be purified from plasma by SPE using an automated
      platform and in-house 96-well SPE protocol (60mg, 3mL; Phenomenex) for extraction of 100 μL
      tissue. All analytes have been previously optimized for extraction efficiencies of between
      80-100% recoveries with CV≤10%. Extracts will be separated and quantified via liquid
      chromatography tandem-MS/MS involving previously published and validated methodologies.
      Briefly, HPLC-ESI-MS/MS analysis will be performed using a SCIEX UPLC coupled 6500 +
      MS/MS-QTRAP™ equipped with an electrospray ionization (ESI) Turbo-V source (blood and fecal
      metabolites), with samples injected onto a Kinetex PFP column and mobile phase gradient
      consisted of 0.1% formic acid (v/v) in water (A) and 0.1% formic acid (v/v) in acetonitrile
      (B), and a 24 min separation time. The targeted metabolite analysis protocol has been
      optimized and validated to detect greater than 200 analytes which are quantified relative to
      authentic commercial and synthetic standards in our library, with putative Phase II
      conjugates identified using fragmentation profiling involving 3-5 unique transitions. This
      involves scanning over 1000 transitions corresponding to sulfate, glucuronide, methyl and
      glycine conjugates. This SMRM methodology was previously optimized for the Rexocyn treatment
      and provides a significantly more sensitive method than traditional multiple reaction
      monitoring (MRM) scanning procedures, as it permits monitoring a greater number of
      transitions per unit of time, in addition to allowing unique compound-dependent parameters to
      be utilized for each analyte, resulting in enhanced sensitivity. Finally, metabolites will be
      confirmed on the basis of retention time (using authentic and synthesized standards where
      possible) and three or more precursor-to-product ion transitions. Seven-point matrix-matched
      calibration curves ranging from 0 to 10 μM will be used for the analysis of flavonoids or
      from 0 to 50 μM for the analysis of phenolic metabolites.

      Intestinal Permeability, Urine Lactulose/Rhamnose:

      Urine sugar concentrations will be determined by high-pressure liquid chromatography. Sucrose
      in 0-5-h urine, lactulose/rhamnose (L/R) ratio in 0-5-h urine, and sucralose/erythritol (S/E)
      ratio in 5-24-h urine will be used as indicators for gastroduodenal, small and large
      intestine permeability, respectively.
    

Intervention name(s): Flavonoid supplement; Placebo
Intervention type(s): Dietary Supplement; Dietary Supplement
Intervention description(s): 2-weeks ingestion of flavonoid supplement; 2-week ingestion of placebo supplement
Allocation: Randomized
Intervention model: Parallel Assignment
Intervention model description: Randomized, parallel group design
Masking: Double (Participant, Investigator)
Primary purpose: Treatment
Keywords: exercise; flavonoids; gut permeability; gut-derived phenolics