Theobromine, Vascular Function and Intestinal apoA-I Production
Condition: Cardiovascular Disease
MeSH terms: Cardiovascular Diseases
Study type: Interventional

Summary: 
      Rationale: Despite successful efforts to lower atherogenic serum low-density lipoprotein
      (LDL) cholesterol concentrations, a substantial residual cardiovascular risk remains. An
      additive strategy to further lower this residual risk may be via raising high-density
      lipoprotein (HDL) concentrations, and in particular those of its major protein constituent
      apolipoprotein A-I (apoA-I). Based on several studies, theobromine may be a promising
      candidate in this respect. Recently, theobromine was shown to increase serum HDL cholesterol
      (HDL-C) concentrations by 0.16 mmol/L or 10% and apoA-I levels with 8%. The question is
      whether this increase in HDL-C and apoA-I concentrations observed translates into an improved
      functionality of the blood vessels. Effects of theobromine on vascular function have never
      been evaluated in a placebo controlled human intervention study.

      Objective: The primary objective is to evaluate the long-term effects of theobromine on
      vascular function in healthy subjects with a slightly lowered HDL-C in the fasting and the
      postprandial state. The second primary objective is to evaluate the long-term effects of
      theobromine on intestinal apoA-I mRNA and protein expression levels in healthy subjects with
      a slightly lowered HDL-C in the fasting and the postprandial state.

      Secondary objectives are to study the long-term effects of theobromine on (1) fasting serum
      HDL-C concentrations, (2) cholesterol efflux capacity and (3) postprandial lipid and glucose
      metabolism.

      Study design: A randomized, double-blind, placebo controlled cross-over design. The total
      study duration will be 12 weeks, consisting of a 4 week experimental period, a 4 week
      wash-out, and a 4 week control period. At the end of the experimental and control periods, a
      postprandial test will take place.

      Study population: Forty-eight healthy men aged 45-70 years and women aged 50-70 years, with
      slightly lowered HDL-C concentrations (men <1.3 mmol/L and women <1.5 mmol/L).

      Intervention: During the experimental period, subjects will consume daily at breakfast an
      drink containing 500 mg theobromine. During the placebo period, the subjects will consume
      daily at breakfast the same drink without theobromine. During the wash-out period, they will
      not consume any of the drinks.

      Main study parameters/endpoints: Measurements will be performed at the end of both 4-week
      intervention periods. The effects of theobromine will be calculated as the absolute
      differences between values obtained at the end of each period. The primary endpoint is the
      change in vascular function defined as % change in flow-mediated dilation (FMD) after intake
      of a daily stressor, a milkshake providing all the three different macronutrients. The second
      primary endpoint is the change in apoA-I mRNA and protein expression on the end of each
      period 5 hours after intake of the milkshake.
    

Detailed description: 
      Rationale: Despite successful efforts to lower atherogenic serum low-density lipoprotein
      (LDL) cholesterol concentrations, a substantial residual cardiovascular risk remains. An
      additive strategy to further lower this residual risk may be via raising high-density
      lipoprotein (HDL) concentrations, and in particular those of its major protein constituent
      apolipoprotein A-I (apoA-I). Based on several studies, theobromine may be a promising
      candidate in this respect. Recently, theobromine was shown to increase serum HDL cholesterol
      (HDL-C) concentrations by 0.16 mmol/L or 10% and apoA-I levels with 8%. The question is
      whether this increase in HDL-C and apoA-I concentrations observed translates into an improved
      functionality of the blood vessels. Effects of theobromine on vascular function have never
      been evaluated in a placebo controlled human intervention study.

      Objective: The primary objective is to evaluate the long-term effects of theobromine on
      vascular function in healthy subjects with a slightly lowered HDL-C in the fasting and the
      postprandial state. The second primary objective is to evaluate the long-term effects of
      theobromine on intestinal apoA-I mRNA and protein expression levels in healthy subjects with
      a slightly lowered HDL-C in the fasting and the postprandial state.

      Secondary objectives are to study the long-term effects of theobromine on (1) fasting serum
      HDL-C concentrations, (2) cholesterol efflux capacity and (3) postprandial lipid and glucose
      metabolism.

      Study design: A randomized, double-blind, placebo controlled cross-over design. The total
      study duration will be 12 weeks, consisting of a 4 week experimental period, a 4 week
      wash-out, and a 4 week control period. At the end of the experimental and control periods, a
      postprandial test will take place.

      Study population: Forty-eight healthy men aged 45-70 years and women aged 50-70 years, with
      slightly lowered HDL-C concentrations (men <1.3 mmol/L and women <1.5 mmol/L).

      Intervention: During the experimental period, subjects will consume daily at breakfast an
      drink containing 500 mg theobromine. During the placebo period, the subjects will consume
      daily at breakfast the same drink without theobromine. During the wash-out period, they will
      not consume any of the drinks.

      Main study parameters/endpoints: Measurements will be performed at the end of both 4-week
      intervention periods. The effects of theobromine will be calculated as the absolute
      differences between values obtained at the end of each period. The primary endpoint is the
      change in vascular function defined as % change in flow-mediated dilation (FMD) after intake
      of a daily stressor, a milkshake providing all the three different macronutrients. The second
      primary endpoint is the change in apoA-I mRNA and protein expression on the end of each
      period 5 hours after intake of the milkshake.

      Nature and extent of the burden and risks associated with participation, benefit and group
      relatedness: Before the study, subjects will be screened twice to determine eligibility
      during two visits of 15 minutes. During these visits, body weight, height and blood pressure
      will be measured. In addition, a venous blood sample (5.5 mL at each occasion) will be drawn.
      During the study, subjects will receive the control and theobromine drinks in random order.
      At day 1 and 14 a fasting venous blood sample (16.5 mL and 7 mL respectively) will be drawn
      and body weight and blood pressure will be measured. At days 25 and 81 again a fasting blood
      sample will be drawn (28 mL). In addition, at day 28 of each experimental period, at t=0
      hours, a fasting venous blood sample (30 mL) will be drawn and vascular function will be
      measured, followed by a 2.5 h postprandial test with again the vascular function
      measurements. As postprandial meal, subjects will receive a high-fat milkshake. Before the
      postprandial test a cannula will be placed and blood will be sampled at t=0, 15, 30, 45, 60,
      90, 120 and 240 (total volume 129.5 mL). Thus, during the entire 13 (screening and study)
      weeks period, in total 444 mL blood will be drawn. Vascular function measurements include a
      panel of non-invasive measurements, i.e. FMD, endo peripheral artherial tonometry (endoPAT),
      pulse wave velocity (PWV) and retinal imaging. Subjects will be asked to fill out a food
      frequency questionnaire two times and to keep a study-diary throughout 12 weeks. On rare
      occasions, blood sampling might cause bruises or hematoma. The vascular function measurements
      are non-invasive and will cause no burden.

      In a subgroup of 10 volunteers, 5 hours after the start of the postprandial test, small
      intestinal biopsies will be taken in the duodenum to determine the difference in intestinal
      apoA-I mRNA and protein expression. Obtaining small intestinal biopsies by standard flexible
      gastroscopy induces local discomfort in the pharynx only during the procedure, which takes
      about 15 minutes, and causes a theoretical risk for perforations (1:1000) and an infection
      risk of 1:1.800.000, with lower risk for healthy subjects. The complication is serious and
      subjects will have to stay in the hospital. If there are serious complaints after the
      gastroscopy (e.g. vomiting with or without blood, or stomach ache) subjects are have to warn
      the project leader or their physician.

      Total time investment for the subjects will be approximately 14.5 hours, and 17.5 hours in
      the subgroup from which biopsies will be taken, excluding travel time.
    

Intervention name(s): Theobromine; Placebo drink
Intervention type(s): Dietary Supplement; Dietary Supplement
Intervention description(s): Theobromine a compound for cocoa
Intervention MeSH terms: Theobromine
Allocation: Randomized
Intervention model: Crossover Assignment
Masking: Quadruple (Participant, Care Provider, Investigator, Outcomes Assessor)
Primary purpose: Prevention
Keywords: Atherosclerosis; Cholesterol; Cholesterol, HDL; Apolipoprotein A-I; Theobromine