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

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

Structurally-related (−)-epicatechin metabolites in humans: assessment using de novo chemically synthesized authentic standards

Accumulating data suggest that diets rich in flavanols and procyanidins are beneficial for human health. In this context, there has been a great interest in elucidating the systemic levels and metabolic profiles at which these compounds occur in humans. While recent progress has been made, there still exist considerable differences and various disagreements with regard to the mammalian metabolites of these compounds, which in turn is largely a consequence of the lack of availability of authentic standards that would allow for the directed development and validation of expedient analytical methodologies. In the present study, we developed a method for the analysis of structurally-related flavanol metabolites using a wide range of authentic standards. Applying this method in the context of a human dietary intervention study using comprehensively characterized and standardized flavanol- and procyanidin-containing cocoa, we were able to identify the structurally-related (−)-epicatechin metabolites (SREM) postprandially extant in the systemic circulation of humans. Our results demonstrate that (−)-epicatechin-3′-β-D-glucuronide, (−)-epicatechin-3′-sulfate, and a 3′-O-methyl(−)-epicatechin-5/7-sulfate are the predominant SREM in humans, and further confirm the relevance of the stereochemical configuration in the context of flavanol metabolism. In addition, we also identified plausible causes for the previously reported discrepancies regarding flavanol metabolism, consisting to a significant extent of inter-laboratory differences in sample preparation (enzymatic treatment and sample conditioning for HPLC analysis) and detection systems. Thus, these findings may also aid in the establishment of consensus on this topic

Regular Consumption of a Flavanol-rich Chocolate can Improve Oxidant Stress in Young Soccer Players

The consumption of a diet rich in certain flavonoids, including the flavanol sub-class, has been associated with a reduced risk for vascular disease. We evaluated the effects of the regular consumption (14 d) of a flavanol-containing milk chocolate (FCMC) or cocoa butter chocolate (CBC) on variables related to vascular disease risk, oxidative stress and physical activity. Twenty-eight free-living, young (18–20 years old) male soccer players consumed daily 105 g of FCMC (168 mg of flavanols) or CBC (<5 mg of flavanols), as part of their normal diet. The consumption of FCMC was significantly associated with a decrease in diastolic blood pressure (-5 mm Hg), mean blood pressure (-5 mm Hg), plasma cholesterol (-11%), LDL-cholesterol (-15%), malondialdehyde (-12%), urate (-11%) and lactate dehydrogenase (LDH) activity (-11%), and an increase in vitamin E/cholesterol (+12%). No relevant changes in these variables were associated with CBC consumption. No changes in the plasma levels of (-)-epicatechin were observed following analysis of fasting blood samples. In conclusion, FCMC consumption was associated with changes in several variables often associated with cardiovascular health and oxidant stress. The presence of significant quantities of flavanols in FCMC is likely to have been one of the contributing factors to these results

Methylxanthines enhance the effects of cocoa flavanols on cardiovascular function: randomized, double-masked controlled studies

Background: Cocoa flavanol intake, especially that of (−)-epicatechin, has been linked to beneficial effects on human cardiovascular function. However, cocoa also contains the methylxanthines theobromine and caffeine, which may also affect vascular function. Objective: We sought to determine whether an interaction between cocoa flavanols and methylxanthines exists that influences cocoa flavanol–dependent vascular effects. Design: Test drinks that contained various amounts of cocoa flavanols (0–820 mg) and methylxanthines (0–220 mg), either together or individually, were consumed by healthy volunteers (n = 47) in 4 different clinical studies—3 with a randomized, double-masked crossover design and 1 with 4 parallel crossover studies. Vascular status was assessed by measuring flow-mediated vasodilation (FMD), brachial pulse wave velocity (bPWV), circulating angiogenic cells (CACs), and blood pressure before and 2 h after the ingestion of test drinks. Results: Although cocoa flavanol intake increased FMD 2 h after intake, the consumption of cocoa flavanols with methylxanthines resulted in a greater enhancement of FMD. Methylxanthine intake alone did not result in statistically significant changes in FMD. Cocoa flavanol ingestion alone decreased bPWV and diastolic blood pressure and increased CACs. Each of these changes was more pronounced when cocoa flavanols and methylxanthines were ingested together. It is important to note that the area under the curve of the plasma concentration of (−)-epicatechin metabolites over time was higher after the co-ingestion of cocoa flavanols and methylxanthines than after the intake of cocoa flavanols alone. Similar results were obtained when pure (−)-epicatechin and the methylxanthines theobromine and caffeine were consumed together. Conclusion: A substantial interaction between cocoa flavanols and methylxanthines exists at the level of absorption, in which the methylxanthines mediate an increased plasma concentration of (−)-epicatechin metabolites that coincides with enhanced vascular effects commonly ascribed to cocoa flavanol intake

Flavan-3-ol-methylxanthine interactions: Modulation of flavan-3-ol bioavailability in volunteers with a functional colon and an ileostomy

Flavan-3-ols, including the flavan-3-ol monomer (-)-epicatechin, are dietary bioactives known to mediate beneficial cardiovascular effects in humans. Recent studies showed that flavan-3-ols could interact with methylxanthines, evidenced by an increase in flavan-3-ol bioavailability with a concomitant increase in flavan-3-ol intake-mediated vascular effects. This study aimed at elucidating flavan-3-ol-methylxanthine interactions in humans in vivo by evaluating the specific contributions of theobromine and caffeine on flavan-3-ol bioavailability. In ileostomists, the effect of methylxanthines on the efflux of flavan-3-ol metabolites in the small intestine was assessed, a parameter important to an understanding of the pharmacokinetics of flavan-3-ols in humans. In a randomized, controlled, triple cross-over study in volunteers with a functional colon (n = 10), co-ingestion of flavan-3-ols and cocoa methylxanthines, mainly represented by theobromine, increased peak circulatory levels (C ) of flavan-3-ols metabolites (+21 ± 8%; p &lt; 0.05). Conversely, caffeine did not mediate a statistically significant effect on flavan-3-ol bioavailability (C = +10 ± 8%, p = n.s.). In a subsequent randomized, controlled, double cross-over study in ileostomists (n = 10), cocoa methylxanthines did not affect circulatory levels of flavan-3-ol metabolites, suggesting potential differences in flavan-3-ol bioavailability compared to volunteers with a functional colon. The main metabolite in ileal fluid was (-)-epicatechin-3'-sulfate, however, no differences in flavan-3-ol metabolites in ileal fluid were observed after flavan-3-ol intake with and without cocoa methylxanthines. Taken together, these results demonstrate a differential effect of caffeine and theobromine in modulating flavan-3-ol bioavailability when these bioactives are co-ingested. These findings should be considered when comparing the effects mediated by the intake of flavan-3-ol-containing foods and beverages and the amount and type of methylxanthines present in the ingested matrixes. Ultimately, these insights will be of value to further optimize current dietary recommendations for flavan-3-ol intake. CLINICAL TRIAL REGISTRATION NUMBER: This work was registered at clinicaltrials.gov as NCT03526107 (study part 1, volunteers with functional colon) and NCT03765606 (study part 2, volunteers with an ileostomy). [Abstract copyright: Copyright © 2023 Elsevier Inc. All rights reserved.

5‐(3′,4′‐Dihydroxyphenyl)‐γ‐Valerolactone is a substrate for human paraoxonase: a novel pathway in flavan‐3‐ol metabolism

Scope Dietary flavan-3-ols are known to mediate cardiovascular benefits. Currently, it is assumed that the levels of flavan-3-ol catabolites detected in humans, 5-(3ʹ,4ʹ-dihydroxyphenyl)-γ-valerolactone (γVL) and 5-(3ʹ,4ʹ-dihydroxyphenyl)-γ-valeric acid (γVA), and their corresponding phase II metabolites, are determined exclusively by the action of the gut microbiome. However, a family of human proteins, paraoxonase (PON), can theoretically hydrolyze γVL metabolites into the corresponding γVAs. This study aims to determine if PON is involved in γVL and γVA metabolism in humans. Methods and results A rapid conversion of γVL into γVA is detected in serum ex vivo (half-life = 9.8 ± 0.3 min) that is catalyzed by PON1 and PON3 isoforms. Phase II metabolites of γVL are also reacted with PON in serum. Following an intake of flavan-3-ol in healthy males (n = 13), the profile of γVA metabolites detected is consistent with that predicted from the reactivity of γVL metabolites with PON in serum. Furthermore, common PON polymorphisms are evaluated to assess the use of γVL metabolites as biomarkers of flavan-3-ol intake. Conclusion PONs are involved in flavan-3-ol metabolic pathway in humans. PON polymorphisms have a minor contribution to inter-individual differences in the levels of γVL metabolites, without affecting their use as a nutritional biomarker

Evaluation at scale of microbiome-derived metabolites as biomarker of flavan-3-ol intake in epidemiological studies.

The accurate assessment of dietary intake is crucial to investigate the effect of diet on health. Currently used methods, relying on self-reporting and food composition data, are known to have limitations and might not be suitable to estimate the intake of many bioactive food components. An alternative are nutritional biomarkers, which can allow an unbiased assessment of intake. They require a careful evaluation of their suitability, including: (a) the availability of a precise, accurate and robust analytical method, (b) their specificity (c) a consistent relationship with actual intake. We have evaluated human metabolites of a microbiome-derived flavan-3-ol catabolite, 5-(3',4'-dihydroxyphenyl)-[gamma]-valerolactone (gVL), as biomarker of flavan-3-ol intake in large epidemiological studies. Flavan-3-ols are widely consumed plant bioactives, which have received considerable interest due to their potential ability to reduce CVD risk. The availability of authentic standards allowed the development of a validated high-throughput method suitable for large-scale studies. In dietary intervention studies, we could show that gVL metabolites are specific for flavan-3-ols present in tea, fruits, wine and cocoa-derived products, with a strong correlation between intake and biomarker (Spearman's r = 0.90). This biomarker will allow for the first time to estimate flavan-3-ol intake and further investigation of associations between intake and disease risk

Impact of polyphenol oxidase on the bioavailability of flavan-3-ols in fruit smoothies: a controlled, single blinded, cross-over study

Flavan-3-ols are bioactive compounds found in a variety of fruits and vegetables (F&V) that have been linked to positive health benefits. Increasing habitual flavan-3-ol intake is challenged by the generally low consumption of F&V. While smoothies are a commonly endorsed, consumer-accepted means to increase the daily intake of these important foods, fruits used for smoothie preparation can have a high polyphenol oxidase (PPO) activity and thus potentially affect the content and bioavailability of flavan-3-ols. To assess whether or not consuming freshly prepared smoothies made with different PPO-containing fruit impacts the bioavailability of the flavan-3-ols, a controlled, single blinded and cross-over study was conducted in healthy men (n = 8) who consumed a flavan-3-ol-containing banana-based smoothie (high-PPO drink), a flavan-3-ol-containing mixed berry smoothie (low-PPO drink) and flavan-3-ols in a capsule format (control). The peak plasma concentration (Cmax) of flavan-3-ol metabolites after capsule intake was 680 ± 78 nmol L−1, which was similar to the levels detected after the intake of the low PPO drink. In contrast, the intake of the high PPO drink resulted in a Cmax of 96 ± 47 nmol L−1, 84% lower than that obtained after capsule intake. In a subsequent study (n = 11), flavan-3-ols were co-ingested with a high-PPO banana drink but contact prior to intake was prevented. In this context, plasma flavan-3-ol levels were still reduced, suggesting an effect possibly related to post-ingestion PPO activity degrading flavan-3-ols in the stomach. There was a substantial range in the PPO activity detected in 18 different fruits, vegetables and plant-derived dietary products. In conclusion, bioavailability of flavan-3-ols, and most likely other dietary polyphenol bioactives, can be reduced substantially by the co-ingestion of high PPO-containing products, the implications of which are of importance for dietary advice and food preparation both at home and in industrial settings

Evaluation of (−)-epicatechin metabolites as recovery biomarker of dietary flavan-3-ol intake

Abstract: Data from dietary intervention studies suggest that intake of (−)-epicatechin mediates beneficial vascular effects in humans. However, population-based investigations are required to evaluate associations between habitual intake and health and these studies rely on accurate estimates of intake, which nutritional biomarkers can provide. Here, we evaluate a series of structurally related (−)-epicatechin metabolites (SREM), particularly (−)-epicatechin-3′-glucuronide, (−)-epicatechin-3′-sulfate and 3′-O-methyl-(−)-epicatechin-5-sulfate (SREMB), as flavan-3-ol and (−)-epicatechin intake. SREMB in urine proved to be a specific indicator of (−)-epicatechin intake, showing also a strong correlation with the amount of (−)-epicatechin ingested (R2: 0.86 (95% CI 0.8l; 0.92). The median recovery of (−)-epicatechin as SREMB in 24 h urine was 10% (IQR 7–13%) and we found SREMB in the majority of participants of EPIC Norfolk (83% of 24,341) with a mean concentration of 2.4 ± 3.2 µmol/L. Our results show that SREMB are suitable as biomarker of (−)-epicatechin intake. According to evaluation criteria from IARC and the Institute of Medicine, the results obtained support use of SREMB as a recovery biomarker to estimate actual intake of (−)-epicatechin

Biomarker-estimated flavan-3-ol intake is associated with lower blood pressure in cross-sectional analysis in EPIC Norfolk

Funder: Mars; doi: http://dx.doi.org/10.13039/100007246Abstract: Flavan-3-ols are a group of bioactive compounds that have been shown to improve vascular function in intervention studies. They are therefore of great interest for the development of dietary recommendation for the prevention of cardio-vascular diseases. However, there are currently no reliable data from observational studies, as the high variability in the flavan-3-ol content of food makes it difficult to estimate actual intake without nutritional biomarkers. In this study, we investigated cross-sectional associations between biomarker-estimated flavan-3-ol intake and blood pressure and other CVD risk markers, as well as longitudinal associations with CVD risk in 25,618 participants of the European Prospective Investigation into Cancer (EPIC) Norfolk cohort. High flavan-3-ol intake, achievable as part of an habitual diet, was associated with a significantly lower systolic blood pressure (− 1.9 (− 2.7; − 1.1) mmHg in men and − 2.5 (− 3.3; − 1.8) mmHg in women; lowest vs highest decile of biomarker), comparable to adherence to a Mediterranean Diet or moderate salt reduction. Subgroup analyses showed that hypertensive participants had stronger inverse association between flavan-3-ol biomarker and systolic blood pressure when compared to normotensive participants. Flavanol intake could therefore have a role in the maintenance of cardiovascular health on a population scale