The metabolome of [2-(14)C](-)-epicatechin in humans: implications for the assessment of efficacy, safety, and mechanisms of action of polyphenolic bioactives

Abstract

Diet is a major life style factor affecting human health, thus emphasizing the need for evidence-based dietary guidelines for primary disease prevention. While current recommendations promote intake of fruit and vegetables, we have limited understanding of plant-derived bioactive food constituents other than those representing the small number of essential nutrients and minerals. This limited understanding can be attributed to some extent to a lack of fundamental data describing the absorption, distribution, metabolism and excretion (ADME) of bioactive compounds. Consequently, we selected the flavanol (-)-epicatechin (EC) as an example of a widely studied bioactive food constituent and investigated the ADME of [2-(14)C](-)-epicatechin (300 μCi, 60 mg) in humans (n = 8). We demonstrated that 82 ± 5% of ingested EC was absorbed. We also established pharmacokinetic profiles and identified and quantified >20 different metabolites. The gut microbiome proved to be a key driver of EC metabolism. Furthermore, we noted striking species-dependent differences in the metabolism of EC, an insight with significant consequences for investigating the mechanisms of action underlying the beneficial effects of EC. These differences need to be considered when assessing the safety of EC intake in humans. We also identified a potential biomarker for the objective assessment of EC intake that could help to strengthen epidemiological investigations.

Trial registration: ClinicalTrials.gov NCT01969994.

Figure 1

(A) Chemical structure of [2-¹⁴C](−)-epicatechin. (B) Schematic representation of study design.

Figure 2

(A) Concentration in whole blood and plasma of [2-¹⁴C](−)-epicatechin -derived radioactivity as a function of time. Data expressed in nM as mean values ± SEM (n = 8). (B) Total [2-¹⁴C](−)-epicatechin -derived radioactivity in urine and feces as a function of time. Data are expressed in μmol as mean values ± SEM (n = 8).

Figure 3. Concentration in plasma of [2-¹⁴C](−)-epicatechin metabolites as a function of time.

SREM: structurally-related (−)-epicatechin metabolites. 5C-RFM: 5-carbon ring fission metabolites. Data are expressed as mean values in nM ± SEM (n = 8). Insert: pie charts depict the relative amount (% of total) of individual SREM and 5C-RFM present in plasma at 1 h, 4 h, 6 h and 12 h after ¹⁴C-EC ingestion (n = 8).

Figure 4. Species-dependent differences in [2-¹⁴C](−)-epicatechin metabolism.

(A) Pie charts depict the relative amount (% of total) of individual SREM present plasma at 30 min post ¹⁴C-EC intake (mouse, rat) and at 1 h post ¹⁴C-EC intake (human). (B) Representative HPLC chromatograms of (yellow) SREM authentic standards (absorbance detection at 280 nm); (green) mouse-specific SREM (on-line radioactivity detection); (red) rat-specific SREM (on-line radioactivity detection); and (blue) human-specific SREM (on-line radioactivity detection).

Figure 5. Schematic representation of (−)-epicatechin metabolism in humans as a function of time post-oral intake.

SREM: structurally related (−)-epicatechin metabolites. 5C-RFM: 5-carbon ring fission metabolites. 3/1C-RFM: 3- and 1-carbon-side chain ring fission metabolites. The structures of the most abundant (−)-epicatechin metabolites present in the systemic circulation and in urine are depicted.