Reliance on self-reports and estimated food composition data in nutrition research introduces significant bias that can only be addressed with biomarkers.

The chemical composition of foods is complex, variable, and dependent on many factors. This has a major impact on nutrition research as it foundationally affects our ability to adequately assess the actual intake of nutrients and other compounds. In spite of this, accurate data on nutrient intake are key for investigating the associations and causal relationships between intake, health, and disease risk at the service of developing evidence-based dietary guidance that enables improvements in population health. Here, we exemplify the importance of this challenge by investigating the impact of food content variability on nutrition research using three bioactives as model: flavan-3-ols, (-)-epicatechin, and nitrate. Our results show that common approaches aimed at addressing the high compositional variability of even the same foods impede the accurate assessment of nutrient intake generally. This suggests that the results of many nutrition studies using food composition data are potentially unreliable and carry greater limitations than commonly appreciated, consequently resulting in dietary recommendations with significant limitations and unreliable impact on public health. Thus, current challenges related to nutrient intake assessments need to be addressed and mitigated by the development of improved dietary assessment methods involving the use of nutritional biomarkers.

Studies about the health benefits of foods or nutrients are often inconsistent. One study may find a health benefit of a particular food and may recommend that people increase their consumption of this food to reduce their disease risk. Yet another study may find the opposite. Inconsistent study results fuel confusion and frustration, and reduce trust in research. Limitations in the studies' designs are likely to be blamed for the inconsistent findings. For example, many studies rely on participants to self-report their food intake and on databases of the nutritional content of food. But people may not accurately report their food intake. Foods vary in their nutritional content, even between two items of the same food such as two apples. And how individuals metabolize foods can further affect the nutrients they receive. Nutritional biomarkers are a potential alternative to measuring dietary intake of specific nutrients. Biomarkers are compounds the body produces when it metabolizes a specific nutrient. Measuring biomarkers therefore give scientists a more accurate and unbiased assessment of nutrient intake. Ottaviani et al. conducted a study to test the differences when estimating nutrient intake using nutritional biomarkers compared with more conventional tools. They analyzed data from a nutrition study that involved over 18,000 participants. The experiments used computer modelling to assess study results using self-reported food intake in combination with food composition database information, or measures of three biomarkers estimating the intake of flavan-3-ols, epicatechin, and nitrates. The models showed that self-reported intake and food database information often led to inaccurate results that did not align well with biomarker measurements. Measuring nutritional biomarkers provides a more accurate and unbiased assessment of nutritional intake. Using these measurements instead of traditional methods for measuring nutrient intake may help increase the reliability of nutrition research. Scientists must work to identify and confirm biomarkers of nutrients to facilitate this work. Using these more precise nutrient measurements in studies may result in more consistent results. It may also lead to more trustworthy recommendations for consumers.

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.

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-(3',4'-dihydroxyphenyl)-γ-valerolactone (γVL) and 5-(3',4'-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.

Dietary flavanols restore hippocampal-dependent memory in older adults with lower diet quality and lower habitual flavanol consumption.

Dietary flavanols are food constituents found in certain fruits and vegetables that have been linked to cognitive aging. Previous studies suggested that consumption of dietary flavanols might specifically be associated with the hippocampal-dependent memory component of cognitive aging and that memory benefits of a flavanol intervention might depend on habitual diet quality. Here, we tested these hypotheses in the context of a large-scale study of 3,562 older adults, who were randomly assigned to a 3-y intervention of cocoa extract (500 mg of cocoa flavanols per day) or a placebo [(COcoa Supplement and Multivitamin Outcomes Study) COSMOS-Web, NCT04582617]. Using the alternative Healthy Eating Index in all participants and a urine-based biomarker of flavanol intake in a subset of participants [n = 1,361], we show that habitual flavanol consumption and diet quality at baseline are positively and selectively correlated with hippocampal-dependent memory. While the prespecified primary end point testing for an intervention-related improvement in memory in all participants after 1 y was not statistically significant, the flavanol intervention restored memory among participants in lower tertiles of habitual diet quality or habitual flavanol consumption. Increases in the flavanol biomarker over the course of the trial were associated with improving memory. Collectively, our results allow dietary flavanols to be considered in the context of a depletion-repletion paradigm and suggest that low flavanol consumption can act as a driver of the hippocampal-dependent component of cognitive aging.

Cocoa Flavanol Supplementation and the Effect on Insulin Resistance in Females Who Are Overweight or Obese: A Randomized, Placebo-Controlled Trial.

There is interest in the impact that dietary interventions can have on preventing the transition from insulin resistance to type 2 diabetes, including a suggestion that the bioactive components of cocoa may enhance fasting insulin sensitivity. However, a role for cocoa flavanols (CF) in reducing insulin resistance in the insulin-stimulated state, an important risk factor for cardiovascular disease, is unresolved. This study investigated whether CF consumption improved whole-body insulin-mediated glucose uptake ('M') in females with overweight/obesity, using a randomized, double-blinded, placebo-controlled, parallel-group design. Thirty-two premenopausal females (19-49 years; 27-35 kg·m-2) with elevated HOMA-IR (HOMA-IR >1.5) supplemented their habitual diet with two servings/day of a high-flavanol cocoa drink (HFC; 609 mg CF/serving; n = 16) or low-flavanol cocoa drink (LFC; 13 mg CF/serving; n = 16) for 4 weeks. Assessment of HOMA-IR and 'M' during a 3-h, 60 mIU insulin·m-2·min-1 euglycemic clamp was performed before and after the intervention. Data are the mean (SD). Changes to HOMA-IR (HFC -0.003 (0.57); LFC -0.0402 (0.86)) and 'M' (HFC 0.99 (7.62); LFC -1.32 (4.88) µmol·kg-1·min-1) after the intervention were not different between groups. Four weeks' consumption of ~1.2 g CF/day did not improve indices of fasting insulin sensitivity or insulin-mediated glucose uptake. A recommendation for dietary supplementation with cocoa flavanols to improve glycemic control is therefore not established.

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 (Cmax) of flavan-3-ols metabolites (+21 ± 8%; p < 0.05). Conversely, caffeine did not mediate a statistically significant effect on flavan-3-ol bioavailability (Cmax = +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).

Measuring the intake of dietary bioactives: Pitfalls and how to avoid them.

Bioactives are food constituents that, while not essential to human life, can affect health. Thus, there is increased interest in developing dietary recommendations for bioactives. Such recommendations require detailed information about the long-term association between habitual intake and health at population scale, and these can only be provided by large-scale observational studies. Nutritional epidemiology relies on the accurate estimation of intake, but currently used methods, commonly based on a 2-step process involving self-reports and food composition tables, are fraught with significant challenges and are unable to estimate the systemic presence of bioactives. Intake assessments based on nutritional biomarkers can provide an advanced alternative, but there are a number of pitfalls that need to be addressed in order to obtain reliable data on intake. Using flavan-3-ols as a case study, we highlight here key challenges and how they may be avoided. Taken together, we believe that the approaches outlined in this review can be applied to a wide range of food constituents, and doing so will improve assessments of the dietary intake of bioactives.

Design and baseline characteristics of participants in the COcoa Supplement and Multivitamin Outcomes Study (COSMOS).

Background Cocoa extract and multivitamins have been proposed to reduce the risk of cardiovascular disease (CVD) and cancer, respectively. However, few randomized clinical trials have tested their long-term effects on these outcomes. Methods The COcoa Supplement and Multivitamin Outcomes Study (COSMOS) is a randomized, double-blind, placebo-controlled, 2 × 2 factorial trial of a cocoa extract supplement and a multivitamin supplement to reduce the risk of CVD and cancer. Here we describe the pragmatic, hybrid design of the trial and baseline characteristics of the trial participants. Results The nationwide study population includes 21,442 U.S. women aged ≥65 years and men aged ≥60 years without baseline myocardial infarction (MI), stroke, or a recent (within the past 2 years) cancer diagnosis. Participants were randomized in a 2 × 2 factorial design to one of four groups: (1) cocoa extract (containing 500 mg/d flavanols, including 80 mg (-)-epicatechin) and a multivitamin (Centrum Silver©); (2) cocoa extract and multivitamin placebo; (3) multivitamin and cocoa extract placebo; or (4) both placebos. Randomization successfully distributed baseline demographic, clinical, behavioral, and dietary characteristics across treatment groups. Baseline biospecimens were collected from 6867 participants, with at least one follow-up biospecimen from 2142 participants. The primary outcome for the cocoa extract intervention is total CVD (a composite of MI, stroke, cardiovascular mortality, coronary revascularization, unstable angina requiring hospitalization, carotid artery surgery, and peripheral artery surgery); the primary outcome for the multivitamin intervention is total invasive cancer. Conclusion COSMOS will provide important information on the health effects of cocoa extract and multivitamin supplementation in older U.S. adults. Clinical Trials Registration: clinicaltrials.gov #NCT02422745.

Validation of a high-throughput method for the quantification of flavanol and procyanidin biomarkers and methylxanthines in plasma by UPLC-MS.

Nutritional biomarkers are critical tools to objectively assess intake of nutrients and other compounds from the diet. In this context, it is essential that suitable analytical methods are available for the accurate quantification of biomarkers in large scale studies. Recently, structurally-related (-)-epicatechin metabolites (SREMs) and 5-(3',4'-dihydroxyphenyl)-γ-valerolactone metabolites (gVLMs) were identified as biomarkers of intake of flavanols and procyanidins, a group of polyphenol bioactives. This study aimed at validating a high throughput method for the quantification of SREMs and gVLMs in plasma along with methylxanthines (MXs), dietary compounds known to interact with flavanol and procyanidin effects. To accomplish this, a full set of authentic analytical standards were used to optimize a micro solid phase extraction method for sample preparation coupled to HPLC-MS detection. Isotopically-labelled standards for all analytes were included to correct potential matrix effects on quantification. Average accuracies of 101%, 93% and 103% were obtained, respectively, for SREMs, gVLMs and MXs. Intra- and inter-day repeatability values were <15%. The method showed linear responses for all analytes (>0.993). Most SREMs and gVLMs had limits of quantifications <5 nM while limits of quantification of MXs were 0.2 µM. All analytes were stable under different tested processing conditions. Finally, the method proved to be suitable to assess SREMs, gVLMs and MXs in plasma collected after single acute and daily intake of cocoa-derived test materials. Overall, this method proved to be a valid analytical tool for high throughput quantification of flavanol and procyanidin biomarkers and methylxanthines in plasma.

Integrated Multi-Omic Analyses of the Genomic Modifications by Gut Microbiome-Derived Metabolites of Epicatechin, 5-(4'-Hydroxyphenyl)-γ-Valerolactone, in TNFalpha-Stimulated Primary Human Brain Microvascular Endothelial Cells.

Cerebral blood vessels are lined with endothelial cells and form the blood-brain barrier. Their dysfunction constitutes a crucial event in the physiopathology of neurodegenerative disorders and cognitive impairment. Epicatechin can improve cognitive functions and lower the risk for Alzheimer's disease or stroke. However, molecular mechanisms of epicatechin on brain vascular endothelium are still unexplored. The objective of this study was to investigate the biological effects of gut microbiome-derived metabolites of epicatechin, 5-(4'-Hydroxyphenyl)-γ-valerolactone-3'-sulfate and 5-(4'-Hydroxyphenyl)-γ-valerolactone-3'-O-glucuronide, in TNF-α-stimulated human brain microvascular endothelial cells at low (nM) concentrations by evaluating their multi-omic modification (expression of mRNA, microRNA, long non-coding RNAs, and proteins). We observed that metabolites are biologically active and can simultaneously modulate the expression of protein-coding and non-coding genes as well as proteins. Integrative bioinformatics analysis of obtained data revealed complex networks of genomics modifications by acting at different levels of regulation. Metabolites modulate cellular pathways including cell adhesion, cytoskeleton organization, focal adhesion, signaling pathways, pathways regulating endothelial permeability, and interaction with immune cells. This study demonstrates multimodal mechanisms of action by which epicatechin metabolites could preserve brain vascular endothelial cell integrity, presenting mechanisms of action underlying epicatechin neuroprotective properties.

Insights into the role of diet and dietary flavanols in cognitive aging: results of a randomized controlled trial.

With the world's population aging, age-related memory decline is an impending cognitive epidemic. Assessing the impact of diet on cognitive aging, we conducted a controlled, randomized, parallel-arm dietary intervention with 211 healthy adults (50-75 years) investigating effects of either a placebo or 260, 510 and 770 mg/day of cocoa flavanols for 12-weeks followed by 8-weeks washout. The primary outcome was a newly-developed object-recognition task localized to the hippocampus' dentate gyrus. Secondary outcomes included a hippocampal-dependent list-learning task and a prefrontal cortex-dependent list-sorting task. The alternative Healthy Eating Index and a biomarker of flavanol intake (gVLM) were measured. In an MRI substudy, hippocampal cerebral blood volume was mapped. Object-recognition and list-sorting performance did not correlate with baseline diet quality and did not improve after flavanol intake. However, the hippocampal-dependent list-learning performance was directly associated with baseline diet quality and improved after flavanol intake, particularly in participants in the bottom tertile of baseline diet quality. In the imaging substudy, a region-of-interest analysis was negative but a voxel-based-analysis suggested that dietary flavanols target the dentate gyrus. While replication is needed, these findings suggest that diet in general, and dietary flavanols in particular, may be associated with memory function of the aging hippocampus and normal cognitive decline.

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

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.

Cocoa Flavanols Improve Endothelial Functional Integrity in Healthy Young and Elderly Subjects.

Cocoa flavanols (CFs) can improve flow-mediated dilation (FMD), blood pressure, and vascular stiffness in healthy subjects. Endothelial microparticles (EMPs) are markers of endothelial functional integrity, reflecting activation and injury. In plasma samples, we investigated whether age-dependent changes in circulating EMPs exist and whether CFs decrease EMPs in healthy humans. The concentrations of CD31+/41-, CD144+, and CD62e+ EMPs (flow cytometry) were increased in healthy elderly (n = 19) compared to young (n = 20) non-smokers. EMPs correlated with age, systolic blood pressure, and pulse wave velocity. CD31+/41- and CD62e+ EMPs inversely correlated with FMD. Following 2 weeks twice-daily CF consumption (450 mg), CD31+/41- and CD144+ EMPs decreased in both young and elderly subjects compared to the CF-free control. The EMP decrease inversely correlated with FMD improvements. Cardiovascular aging is associated with increased EMPs that can be modulated by dietary flavanols along with improvements in vascular function. This indicates that flavanol consumption can improve endothelial functional integrity in healthy humans.

(-)-Epicatechin metabolites promote vascular health through epigenetic reprogramming of endothelial-immune cell signaling and reversing systemic low-grade inflammation.

Ingestion of (-)-epicatechin flavanols reverses endothelial dysfunction by increasing flow mediated dilation and by reducing vascular inflammation and oxidative stress, monocyte-endothelial cell adhesion and transendothelial monocyte migration in vitro and in vivo. This involves multiple changes in gene expression and epigenetic DNA methylation by poorly understood mechanisms. By in silico docking and molecular modeling we demonstrate favorable binding of different glucuronidated, sulfated or methylated (-)-epicatechin metabolites to different DNA methyltransferases (DNMT1/DNMT3A). In favor of this model, genome-wide DNA methylation profiling of endothelial cells treated with TNF and different (-)-epicatechin metabolites revealed specific DNA methylation changes in gene networks controlling cell adhesion-extravasation endothelial hyperpermeability as well as gamma-aminobutyric acid, renin-angiotensin and nitric oxide hypertension pathways. Remarkably, blood epigenetic profiles of an 8 weeks intervention with monomeric and oligomeric flavanols (MOF) including (-)-epicatechin in male smokers revealed individual epigenetic gene changes targeting similar pathways as the in vitro exposure experiments in endothelial cells. Furthermore, epigenetic changes following MOF diet intervention oppose atherosclerosis associated epigenetic changes. In line with biological data, the individual epigenetic response to a MOF diet is associated with different vascular health parameters (glutathione peroxidase 1 and endothelin-1 expression, acetylcholine-mediated microvascular response), in part involving systemic shifts in blood immune cell types which reduce the neutrophil-lymphocyte ratio (NLR). Altogether, our study suggests that different (-)-epicatechin metabolites promote vascular health in part via epigenetic reprogramming of endothelial-immune cell signaling and reversing systemic low-grade inflammation.

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

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.

Assessing the respective contributions of dietary flavanol monomers and procyanidins in mediating cardiovascular effects in humans: randomized, controlled, double-masked intervention trial.

Background: Flavanols are an important class of food bioactives that can improve vascular function even in healthy subjects. Cocoa flavanols (CFs) are composed principally of the monomer (-)-epicatechin (∼20%), with a degree of polymerisation (DP) of 1 (DP1), and oligomeric procyanidins (∼80%, DP2-10). Objective: Our objective was to investigate the relative contribution of procyanidins and (-)-epicatechin to CF intake-related improvements in vascular function in healthy volunteers. Design: In a randomized, controlled, double-masked, parallel-group dietary intervention trial, 45 healthy men (aged 18-35 y) consumed the following once daily for 1 mo: 1) a DP1-10 cocoa extract containing 130 mg (-)-epicatechin and 560 mg procyanidins, 2) a DP2-10 cocoa extract containing 20 mg (-)-epicatechin and 540 mg procyanidins, or 3) a control capsule, which was flavanol-free but had identical micro- and macronutrient composition. Results: Consumption of DP1-10, but not of either DP2-10 or the control capsule, significantly increased flow-mediated vasodilation (primary endpoint) and the concentration of structurally related (-)-epicatechin metabolites (SREMs) in the circulatory system while decreasing pulse wave velocity and blood pressure. Total cholesterol significantly decreased after daily intake of both DP1-10 and DP2-10 as compared with the control. Conclusions: CF-related improvements in vascular function are predominantly related to the intake of flavanol monomers and circulating SREMs in healthy humans but not to the more abundant procyanidins and gut microbiome-derived CF catabolites. Reduction in total cholesterol was linked to consumption of procyanidins but not necessarily to that of (-)-epicatechin. This trial was registered at clinicaltrials.gov as NCT02728466.

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.

Use of LC-MS for the quantitative analysis of (poly)phenol metabolites does not necessarily yield accurate results: Implications for assessing existing data and conducting future research.

Plant-derived, dietary (poly)phenols have potential effects on disease-risk reduction and primary disease prevention. The characterization of (poly)phenol absorption, distribution, metabolism and excretion (ADME) is recognized as crucial step to further advance nutritional and biomedical research of these compounds; and given that (poly)phenols are extensively metabolized after ingestion, accurate assessments of their in vivo metabolites is required. It has become common practice to use unmetabolized parent compounds as reference standards when quantifying (poly)phenol metabolites by LC-MS, although little is known about the accuracy of this approach. To investigate this situation with routinely used LC-MS conditions, the signal yielded by the flavan-3-ol (-)-epicatechin was compared to those of authentic standards of its phase II and microbiota-derived metabolites. The results obtained revealed underestimations up to 94% and overestimations up to 113% of individual epicatechin metabolites. Inaccurate quantitative estimates were also obtained when phase II metabolites of other (poly)phenols were quantified by reference to their unmetabolized parent compounds. This demonstrates the importance of using structurally-identical authentic metabolites as reference compounds when quantifying (poly)phenol metabolites by LC-MS. This is of importance, not just to the accuracy of ADME studies, but for the identification and validation of (poly)phenol metabolites as biomarkers of intake in epidemiological studies.

Recommending flavanols and procyanidins for cardiovascular health: Revisited.

The last 8 years have seen significant developments in our understanding of dietary flavanols and procyanidins in the context of human health and nutrition. During the same time, recognition of the importance of nutrition in primary disease prevention and health maintenance has increased. In addition, the concept of dietary bioactives (food constituents that although not essential to human life and procreation, may nevertheless play an important role in disease risk reduction, primary disease prevention, and healthy aging) has been created and developed. Applying assessment criteria specific to health maintenance and primary disease prevention, we aimed at broadly evaluating and discussing currently available data on flavanols and procyanidins, with an eye towards potentially advancing the future development of dietary guidelines and public health recommendations. Novel insights and advancements as well as current gaps and shortcomings in our understanding are identified and discussed. While centered on flavanols and procyanidins, the outcomes of this review may also have broader relevance for the further development of the concept of bioactives, and any future framework for the assessment of their role in human health and nutrition.

Absorption, metabolism, distribution and excretion of (-)-epicatechin: A review of recent findings.

This paper reviews pioneering human studies, their limitations and recent investigations on the absorption, metabolism, distribution and excretion (aka bioavailability) of (-)-epicatechin. Progress has been made possible by improvements in mass spectrometric detection when coupled to high performance liquid chromatography and through the increasing availability of authentic reference compounds of in vivo metabolites of (-)-epicatechin. Studies have shown that [2-14C](-)-epicatechin is absorbed in the small intestine with the 12 structural-related (-)-epicatechin metabolites (SREMs), mainly in the form of (-)-epicatechin-3'-O-glucuronide, 3'-O-methyl-(-)-epicatechin-5-sulfate and (-)-epicatechin-3'-sulfate, attaining sub-µmol/L peak plasma concentrations (Cmax) ∼1 h after ingestion. SREMs were excreted in urine over a 24 h period in amounts corresponding to 20% of (-)-epicatechin intake. On reaching the colon the flavan-3-ol undergoes microbiota-mediated conversions yielding the 5C-ring fission metabolites (5C-RFMs) 5-(hydroxyphenyl)-γ-valerolactones and 5-(hydroxyphenyl)-γ-hydroxyvaleric acids which appear in plasma as phase II metabolites with a Cmax of 5.8 h after intake and are excreted in quantities equivalent to 42% of the ingested (-)-epicatechin. Other catabolites excreted in 0-24 h urine in amounts equivalent to 28% of intake included 3-(3'-hydroxyphenyl)hydracrylic acid, hippuric acid and 3'-hydroxyhippuric acid. Overall (-)-epicatechin is highly bioavailable with urinary excretion indicating that 95% is absorbed and passes through the circulatory systems as a diversity of phase II metabolites. Rats produce a very different profile of SREMs than that of humans. These findings demonstrate that ex vivo studies investigating the mechanisms underlying the protective effects of (-)-epicatechin on human health should make use of physiological concentrations human of SREMs and 5C-RFMs, and not the parent (-)-epicatechin, with model systems derived from human cells. In epidemiological studies 5-(4'-hydroxyphenyl)-γ-valerolactone-3'-sulfate and 5-(4'-hydroxyphenyl)-γ-valerolactone-3'-O-glucuronide, the principal 5C-RFMs in both plasma and urine, could serve as key biomarkers of (-)-epicatechin intake.