Influence of Flavonoids and Caffeine Supplementation on Resting Metabolism
Condition: Diet Modification
Study type: Interventional

Summary: 
      The purpose of this study is to compare the effect of ingesting caffeine and mixed flavonoids
      (4 capsules, split between breakfast and lunch) on energy expenditure and fat oxidation in a
      metabolic chamber with 20 women (non-obese, healthy, ages 20-47 years, pre-menopausal). We
      hypothesize that based on the existing literature, ingestion of a double dose of the
      caffeine-mixed flavonoid supplement compared to placebo will increase fat oxidation and
      increase 24 h energy expenditure by about 75 kilocalories.
    

Detailed description: 
      This study will utilize a randomized, double-blind, cross-over design, comparing acute
      ingestion of the mixed flavonoid supplement (MF) compared to placebo control (PC) in healthy,
      non-obese, pre-menopausal women. The study will consist of two 24-hour study periods spent in
      the recently renovated indirect room calorimeter (i.e., metabolic chamber) at the University
      of North Carolina Chapel Hill Nutrition Research Institute (UNC NRI). The 24-hour study
      periods in the metabolic chamber will be 4 weeks apart. Primary outcome measures will be
      24-hour energy expenditure (EE), resting metabolic rate (RMR), sleeping metabolic rate (SMR),
      substrate utilization from the respiratory quotient (RQ), and physical activity energy
      expenditure (AEE).

      Following recruitment and eligibility assessment, study participants will be randomly
      assigned to complete either the mixed flavonoid (MF) or the placebo control (PC) study day.
      After a 4-week washout, study participants will cross over and complete the alternative
      treatment. A method of randomly permuted blocks will be generated using web-based
      randomization software (www.randomization.com) resulting in 10 study participants receiving
      MF during the first week, and 10 study participants receiving the PC during the first week.
      Study participants will consume 2 MF or PC capsules 30 min before breakfast, and 2 MF or PC
      capsules 30 min before lunch. During both study days, study participants will be fed in
      energy balance (i.e., energy intake will be matched to energy expenditure).

      2. Experimental Study Participants: Using data generated previously in the UNC NRI metabolic
      chamber, a sample size calculation with 80% power revealed that 15-20 study participants
      would be needed to detect a 50 kcal difference in 24-h EE. To account for attrition, 25 study
      participants will be recruited via mass advertisement throughout the local area.
      Pre-menopausal women will be chosen because this is a target population of the sponsor.

      3. Pre-Study Baseline Testing Eligibility will be determined in the outpatient clinical suite
      at the UNC NRI. Body composition (fat mass and fat free mass (FFM)) will be determined via
      dual energy x-ray absorptiometry (DXA) (GE Lunar iDXA; Milwaukee, WI). Body mass index (BMI,
      kg/m2) will be calculated for measured height and weight. Resting metabolic rate (RMR) will
      be estimated using a FFM-based equation [418 + (20.3 FFM)] (J Appl Physiol
      1993;75:2514-2520). This estimated RMR will be used to calculate total dietary energy intake
      while in the metabolic chamber: RMR x physical activity level (PAL) of 1.3, and then adjusted
      using measured data (details provided below). A small blood sample will be obtained at this
      time and T3, T4, and TSH levels assessed through the clinical lab at the Lab Corp
      (Burlington, NC). A urine pregnancy test will be performed.

      4. Indirect calorimetry: The metabolic chamber at the Nutrition Research Institute in
      Kannapolis, NC is an open-circuit, whole room indirect calorimeter. The CO2 and O2 analysers
      are differential, with full scale readings set for 0%-1%. O2 consumption, CO2 production, EE
      and RQ are recorded each minute. EE is calculated using an abbreviated Weir's formula (VO2 X
      3.941) + (VCO2 X 1.106), where VO2 is the volume of oxygen consumed in L/minute and VCO2 is
      the volume of carbon dioxide released in L/minute. RQ is calculated as VCO2/VO2. Area under
      the curve (AUC) will be calculated using RQ data for the four hours following breakfast,
      lunch and dinner as well as sleeping hours between midnight and 6:00 am. Spontaneous physical
      activity will be measured each minute using a total room sensor. To calculate resting
      metabolic rate (RMR), EE will be plotted against the activity motion sensor output (each
      averaged over 30 minutes), and the y-intercept of the linear regression taken as EE in the
      inactive state. RMR will be multiplied by 1440 minutes to extrapolate to 24 hours.
      Twenty-four hour sleeping metabolic rate (SMR) will be determined as the lowest mean EE
      (kJ/minute) measured over 3 consecutive hours between midnight and 6:30 am and multiplied by
      1440 minutes. Diet induced thermogenesis (DIT) will be calculated by subtraction of SMR from
      RMR. Activity induced EE (AEE) will be calculated as the difference between 24 hour EE and
      RMR.

      5. Metabolic chamber study day protocol. Subjects will arrive at the Nutrition Research
      Institute Building (500 Laureate Way) at 7:00 am. A urine pregnancy test will be performed.
      At 7:30 am, study participants will report to the metabolic chamber following an overnight
      fast (no food or beverages containing calories, alcohol, or caffeine from 11 pm). Study
      participants will be instructed on expectations of their stay and weighed in scrubs without
      shoes. At 8:00 am study participants will be sealed in the chamber. Except for a 2-minute
      interval each hour during which study participants will be requested to stand and stretch,
      study participants will remain seated or reclined, but awake throughout the day. Study
      participants will be asked to perform necessary daily activities during these 2-minute
      intervals. Breakfast (9:00 am), lunch (1:30 pm), and dinner (7:00 pm), will be served through
      an air-lock passage. Meals will be completed within 30 minutes of serving. Two MF capsules or
      placebo will be consumed 30 min before breakfast, and then again 30 minutes before lunch. At
      10:30 pm, study participants will be asked to lie down for sleep. Study participants will be
      awakened at 6:30 am and allowed to move about the chamber to gather their belongings. At 7:15
      am study participants will exit the chamber and be weighed.

      6. Design of Metabolic Diets: Eucaloric diets will be designed to provide approximately 35%
      fat, 49% carbohydrates and 16% protein, reflecting current recommendations for this
      population group. Menus will be designed using a nutrient calculation and food management
      software, and consist of bagel, peanut butter, apple juice, whole-wheat bread, turkey,
      cheese, mayonnaise, buttery spread (10% kcal as fat), potato chips, lasagna, carrots,
      broccoli, rolls, and muffin. No beverages or foods containing caffeine will be served. The
      same foods will be served at both chamber visits. A baseline menu for each subject will be
      prepared based on calculated RMR X 1.3, reflecting the sedentary nature of the study day. To
      ensure energy balance conditions, the baseline menu of the first visit will be modified
      according to measured EE data at 3 hours (includes breakfast) and 7 hours (includes breakfast
      and lunch). Subsequent meals will be adjusted accordingly with 100 kcal peach muffins
      containing the same proportion of fat, carbohydrate and protein as the meals. Study
      participants will be fed an identical amount of the same meal at their second visit.

      7. Statistical Analysis: Data will be analyzed using SAS (Cary, NC). To guard against any
      carryover effect from visit 1 to visit 2, a repeated measures regression with an unstructured
      correlation matrix within subject will be run with treatment (MF and PC) and visit (1 and 2)
      in the model along with an interaction term. If the interaction term is non-significant, then
      it will be removed and the model re-run. Two energy expenditure curves, one for the MF day,
      and one for the PC day, will be generated for each subject with the x axis representing time
      (min), and the y axis representing energy expenditure (kcal). The area under the energy
      expenditure curve for defined blocks of time will be calculated by using the trapezoid rule
      in the EXPAND procedure in SAS (SAS Institute, Inc., Cary, NC). A paired t-test on the
      log-transformed area will be performed to compare the energy expenditure of each period in
      the MF day with the corresponding period in the PC day. The Shapiro-Wilk test in the
      UNIVARIATE procedure in SAS will be used for normality check. The Benjamini-Hochberg method
      for false discovery rate correction in the MULTTEST procedure in SAS will be used for
      multiple testing corrections.

      SUPPLEMENT:

      The mixed flavonoid supplement and placebo capsules will look identical and be supplied by
      Reoxcyn Discoveries Group LLC (Salt Lake City, UT). Active ingredients in the flavonoid
      capsules include vitamin C, wild bilberry fruit extract, green tea leaf extract, quercetin,
      caffeine, and omega 3 fatty acids (see Appendix B). Capsule fill ingredients include Nu-Flow
      70R (from powdered rice hulls), tapioca from cassava root, natural bamboo silica, and
      marshmallow root. Placebo capsules will contain only the fill ingredients (without the active
      ingredients). Two MF and PC capsules will be consumed 30 minutes before breakfast, and then
      again before lunch. The MF capsules will provide 658 mg flavonoids and 214 mg caffeine.
    

Intervention name(s): Flavonoid, caffeine; Placebo
Intervention type(s): Dietary Supplement; Dietary Supplement
Intervention description(s): 2 flavonoid/caffeine capsules before breakfast, 2 capsules before lunch; 2 placebo capsules before breakfast, 2 capsules before lunch
Intervention MeSH terms: Caffeine
Allocation: Randomized
Intervention model: Crossover Assignment
Intervention model description: Randomized, crossover
Masking: Double (Participant, Investigator)
Primary purpose: Treatment
Keywords: energy expenditure; caffeine; flavonoids; fat metabolism