The Influence of the Microbiome on the Pharmacokinetics of Flavan-3-ols
Condition: Diet Modification
Study type: Interventional

Summary: 
      Cocoa beans are of major interest due to their various beneficial health effects, indicated
      to be caused by its cocoa flavan-3-ols. (-)-Epicatechin is the most abundant flavan-3-ol in
      these cocoa beans. Its metabolization in the colon results in bioactive valerolactone and
      valeric acid metabolites and derivatives after phase II metabolism. Interindividual
      differences in health effects following (-)-epicatechin consumption are observed, which are
      suggested to be caused by large interindividual differences in bioavailable metabolite
      concentrations. As the colonic microbiota is responsible for the metabolization of ~70% of
      total (-)-epicatechin intake, and ~42% of total (-)-epicatechin intake leads to valerolactone
      and valeric acid metabolites, it is hypothesized that the large interindividual variation in
      microbial gut composition is responsible for the heterogeneity in metabolite concentration
      and in its subsequent health effects. Furthermore, individuals can be stratified into two
      pharmacotypes, slow and fast microbial metabolizers, which can produce metabolites at
      different rates.

      The aim of this single-arm study is to investigate if the microbial composition in the gut
      determines the rate and extent of metabolization, following an acute consumption of about
      160mg of pure (-)-epicatechin. The pharmacokinetics of the (-)-epicatechin metabolites will
      be followed in plasma over 48h with a focus on the first fifteen hours and potentially in
      urine over 24h. Valerolactone and valeric acid metabolite profiles in plasma and urine will
      be obtained by Q-TOF-LC-MS. The microbial fingerprint of each individual will be obtained via
      DNA extraction, flow cytometry and 16s rRNA sequencing of fecal samples.
    

Detailed description: 
      The aim of this study is to investigate if the microbial composition in the gut can determine
      the rate and extent of valerolactone and valeric acid production following an acute
      consumption of 160mg of pure (-)-epicatechin. The gut microbial composition of each
      participant will be linked with its metabolite plasma profile to investigate if the large
      interindividual variation in metabolite formation can be explained by the variation in
      microbial composition in the colon. This knowledge might contribute to a better understanding
      of the correlation between epicatechin intake and its observed beneficial, but at the same
      time very variable health effects.

      This single-arm trial lasts a total of five days, with one day (day 3) on site at the
      laboratory (UZ Ghent, Department of Rehabilitation Sciences). Two days prior to the test day,
      subjects will be instructed to follow a low-flavan-3-ol diet for the next five days, until
      48h after the supplement intake, to establish a low dietary background of flavan-3-ols. A
      list of restrictions on foods and beverages rich in flavan-3-ols, such as chocolate, tea, red
      wine and some fruit and vegetables, will be provided. Moreover, a standardized
      flavan-3-ol-free meal and snack will be provided to consume the evening prior to the test
      day, together with at least 0.5L of water. One day prior to the test day, each participant
      will collect a stool sample for the determination of their microbial fingerprint. Stool
      collection material will be provided to the subjects on beforehand. In addition, no strenuous
      exercise can be performed on this day.

      On the test day, participants arrive at the laboratory around 8am after an overnight fast of
      minimum 8h and maximum 14h. This day lasts approximately 15h, until 11pm. First, a
      questionnaire will be completed to evaluate compliance with the instructed diet and to check
      the general well-being of the participants. Second, a medical doctor will insert a catheter
      in the median cubital vein in the forearm, after which a first blood sample will be drawn.
      Third, participants will be asked to empty the bladder, have breakfast and take the
      epicatechin supplement within ten minutes after breakfast. Standardized flavan-3-ol-free
      breakfast, lunch, dinner and three snacks will be offered at scheduled time points throughout
      the day. Water is ad libitum with a minimum of 1.5L. Lastly, a food frequency questionnaire
      will be completed.

      Each hour after supplement intake, venous blood will be drawn in vacutainers until fifteen
      samples of 4mL are collected (0h-14h). On time points 0, 2, 6, 8 and 12h, additional blood
      samples will be drawn in serum vacutainers. Urine will be pooled in three timeframes (0-6h,
      6-12h and 12-24h). When all blood samples are collected, the catheter will be removed and a
      questionnaire will be completed to evaluate the test day and well-being of the participants.
      On each of the two following mornings (24h and 48h), one additional blood sample will be
      taken after an overnight fast. The pooled urine (12-24h) will be collected 24h after
      supplement intake. Some participants will also conduct this trial without the (-)-epicatechin
      supplement to verify the effectiveness of the flavan-3-ol-free diet .

      Valerolactone and valeric acid metabolite profiles will be obtained by Quadrupole Time of
      Flight Liquid chromatography-mass spectrometry (Q-TOF-LC-MS). The microbial fingerprint of
      each individual will be obtained via DNA extraction, flow cytometry and 16s rRNA sequencing
      of fecal samples.
    

Intervention name(s): (-)-Epicatechin
Intervention type(s): Dietary Supplement
Intervention description(s): Administration of a capsule with 150 mg of pure (-)-epicatechin after 48h low-flavanol diet.
Intervention model: Single Group Assignment
Intervention model description: Single-dose study where all participants consume one epicatechin supplement.
Masking: None (Open Label)
Primary purpose: Basic Science
Keywords: Valerolactones; (-)-Epicatechin; Microbiota; Pharmacokinetics; Interindividual differences; Q-TOF-LC-MS