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.

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.

There is no direct competition between arginase and nitric oxide synthase for the common substrate l-arginine.

AIMS: Extrahepatic arginases are postulated to be involved in cardiovascular-related pathologies by competing with nitric oxide synthase (NOS) for the common substrate l-arginine, subsequently decreasing nitric oxide production. However, previous models used to study arginase and NOS competition did not account for steady state level of l-arginine pool, which is dependent on conditions of l-arginine supply and utilization pathways. This work aimed at revisiting the concept of NOS and arginase competition while considering different conditions of l-arginine supply and l-arginine utilization pathways. METHODS AND RESULTS: Mouse macrophage-like RAW cells and human vascular endothelial cells co-expressing NOS and arginase were used to reevaluate the concept of substrate competition between arginase and NOS under conditions of l-arginine supply that mimicked either a continuous (similar to in vivo conditions) or a limited supply (similar to previous in vitro models). Enzyme kinetics simulation models were used to gain mechanistic insight and to evaluate the tenability of a substrate competition between the two enzymes. In addition to arginase and NOS, other l-arginine pathways such as transporters and utilization towards protein synthesis were considered to understand the intricacies of l-arginine metabolism. Our results indicate that when there is a continuous supply of l-arginine, as is the case for most cells in vivo, arginase does not affect NOS activity by a substrate competition. Furthermore, we demonstrate that l-arginine pathways such as transporters and protein synthesis are more likely to affect NOS activity than arginase. CONCLUSIONS: Arginase does not outcompete NOS for the common substrate l-arginine. Findings from this study should be considered to better understand the role of arginase in certain pathologies and for the interpretation of in vivo studies with arginase inhibitors.

Genetic modification of flavone biosynthesis in rice enhances biofilm formation of soil diazotrophic bacteria and biological nitrogen fixation.

Improving biological nitrogen fixation (BNF) in cereal crops is a long-sought objective; however, no successful modification of cereal crops showing increased BNF has been reported. Here, we described a novel approach in which rice plants were modified to increase the production of compounds that stimulated biofilm formation in soil diazotrophic bacteria, promoted bacterial colonization of plant tissues and improved BNF with increased grain yield at limiting soil nitrogen contents. We first used a chemical screening to identify plant-produced compounds that induced biofilm formation in nitrogen-fixing bacteria and demonstrated that apigenin and other flavones induced BNF. We then used CRISPR-based gene editing targeting apigenin breakdown in rice, increasing plant apigenin contents and apigenin root exudation. When grown at limiting soil nitrogen conditions, modified rice plants displayed increased grain yield. Biofilm production also modified the root microbiome structure, favouring the enrichment of diazotrophic bacteria recruitment. Our results support the manipulation of the flavone biosynthetic pathway as a feasible strategy for the induction of biological nitrogen fixation in cereals and a reduction in the use of inorganic nitrogen fertilizers.

Structurally related (-)-epicatechin metabolites and gut microbiota derived metabolites exert genomic modifications via VEGF signaling pathways in brain microvascular endothelial cells under lipotoxic conditions: Integrated multi-omic study.

Dysfunction of blood-brain barrier formed by endothelial cells of cerebral blood vessels, plays a key role in development of neurodegenerative disorders. Epicatechin exerts vasculo-protective effects through genomic modifications, however molecular mechanisms of action, particularly on brain endothelial cells, are largely unknow. This study aimed to use a multi-omic approach (transcriptomics of mRNA, miRNAs and lncRNAs, and proteomics), to provide novel in-depth insights into molecular mechanisms of how metabolites affect brain endothelial cells under lipid-stressed (as a model of BBB dysfunction) at physiological concentrations. We showed that metabolites can simultaneously modulate expression of protein-coding, non-coding genes and proteins. Integrative analysis revealed interactions between different types of RNAs and form functional groups of genes involved in regulation of processing like VEGF-related functions, cell signaling, cell adhesion and permeability. Molecular modeling of genomics data predicted that metabolites decrease endothelial cell permeability, increased by lipotoxic stress. Correlation analysis between genomic modifications observed and genomic signature of patients with vascular dementia and Alzheimer's diseases showed opposite gene expression changes. Taken together, this study describes for the first time a multi-omic mechanism of action by which (-)-epicatechin metabolites could preserve brain vascular endothelial cell integrity and reduce the risk of neurodegenerative diseases. SIGNIFICANCE: Dysfunction of the blood-brain barrier (BBB), characterized by dysfunction of endothelial cells of cerebral blood vessels, result in an increase in permeability and neuroinflammation which constitute a key factor in the development neurodegenerative disorders. Even though it is suggested that polyphenols can prevent or delay the development of these disorders, their impact on brain endothelial cells and underlying mechanisms of actions are unknow. This study aimed to use a multi-omic approach including analysis of expression of mRNA, microRNA, long non-coding RNAs, and proteins to provide novel global in-depth insights into molecular mechanisms of how (-)-epicatechin metabolites affect brain microvascular endothelial cells under lipid-stressed (as a model of BBB dysfunction) at physiological relevant conditions. The results provide basis of knowledge on the capacity of polyphenols to prevent brain endothelial dysfunction and consequently neurodegenerative disorders.

Absorption, distribution, metabolism and excretion of apigenin and its glycosides in healthy male adults.

The bioavailability of apigenin and its O-glycosides in humans was investigated with apigenin-4'-glucuronide (Ap-4'-GlcUA), apigenin-7-glucuronide and apigenin-7-sulfate being identified as in vivo metabolites. Apigenin per se was poorly absorbed with metabolites equivalent to 0.5% of intake excreted in urine 0-24 h post-intake. Consumption of a parsley drink containing apigenin-7-O-(2″-O-apiosyl)glucoside resulted in the peak plasma concentration (Cmax) of Ap-4'-GlcUA occurring after 4 h, indicative of absorption in the lower gastrointestinal tract (GIT). Urinary excretion of the three metabolites corresponded to 11.2% of intake. Ingestion of dried powdered parsley leaves with yogurt extended the Cmax of Ap-4'-GlcUA to 6 h. Consumption of chamomile tea containing apigenin-7'-O-glucoside resulted in a 2 h Cmax of Ap-4'-GlcUA, in keeping with absorption in the upper GIT. Urinary excretion was equivalent to 34% of intake. Intake of the parsley drink provided information on intra- and inter-individual variations in the level of excretion of the apigenin metabolites. CLINICAL TRAIL REGISTRATION NUMBER: This trail was registered at clinicaltrials.gov as NCT03526081.

Development and validation of HPLC-MS2 methodology for the accurate determination of C4-C8 B-type flavanols and procyanidins.

Cocoa flavanols and procyanidins (CFs), natural dietary bioactives, have been studied extensively over the past two decades for their potential health benefits. Research on their safety and efficacy is critically dependent upon on the ability to reliably characeterize the research materials that are utilized, and with growing consumer availability of CF-based products, reliable methods for the detection of potential adulteration are of increasing importance. This research focused on the development of a high performance liquid chromatography-tandem mass spectrometry method (HPLC-MS2) using primary standards and 13C-labelled procyanidins as internal standards. The ability of MS2 detection to discriminate A- and B-type procyanidins was demonstrated. Method performances were validated for degrees of polymerization up to four in seven model food matrices. Accuracy ranged from 90.9 to 125.4% and precision was < 10% at lower concentrations. Finally, the method was applied to cocoa-based samples and compared to the AOAC 2020.05 analytical protocol, supporting the use of NIST 8403 as reference material for HPLC-MS2 analysis.

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.

Evolution of cocoa flavanol analytics: impact on reporting and cross-study comparison.

Cocoa flavanols (CF) are a group of dietary bioactives that have been studied for their potential health benefits for over two decades. In this time, multiple methods for CF testing have evolved, introducing the potential for differences in reported CF content. The reliable characterization of CF content in food and test materials used in clinical studies is critical to comparisons of research studies over time, as well as critical to enabling the systematic reviews and meta-analyses required to support dietary recommendations of bioactives. In this work, we compared two analytical methods that have been widely applied to characterize materials used in clinical research and a method newly recognized by AOAC as the official method for CF analysis. Differences in accuracy of -36% to +20% were observed when comparing CF contents determined with these methods, supporting the notion that CF values determined across methods are not directly comparable. To address differences, a linear regression model was developed to predict CF values. This approach was cross-validated and directly applied to the conversion of CF values published in key scientific papers on the benefits of CF. This work provides a valid tool to compare CF values reported across these different methods and enables comparisons and interpretation of studies investigating the bioactivity of CF.

Single-Laboratory Validation for the Determination of Cocoa Flavanols and Procyanidins (by Degree of Polymerization DP1-7) in Cocoa-Based Products by Hydrophilic Interaction Chromatography Coupled with Fluorescence Detection: First Action 2020.05.

BACKGROUND: Flavanols and procyanidins are complex bioactives found in many foods such as cocoa. As their consumption is associated with health benefits, cocoa flavanols and procyanidins are receiving increasing attention from consumers, industry, researchers, and regulators. OBJECTIVE: The objective of this study is to validate a method using hydrophilic interaction chromatography (HILIC) with fluorescence detection (FLD) and a commercially available reference material for the determination of flavanols and procyanidins (CF) in cocoa-based products. METHODS: Method performances were evaluated for cocoa matrices with CF content that ranged from 0.8 to 500 mg/g, which included low CF matrices (milk and dark chocolate, cocoa powder, and liquor) and high CF matrices (cocoa extract and dietary supplement products). The method was validated in a single-laboratory by determining sensitivity, selectivity, linearity, stability, robustness, accuracy, and precision for each of the matrices. RESULTS: The method was validated for cocoa matrices with CF content that ranged from 0.8 to 500 mg/g. Accuracy ranged from 86 to 99% and repeatability (RSDr) from 1.5 to 8.6% for CF. CONCLUSIONS: Analytical performances acquired through this single-laboratory validation study for a wide range of cocoa-based matrices demonstrate that this method is fit-for-purpose for the determination of flavanols and procyanidins in cocoa-based products. HIGHLIGHTS: Hydrophilic interaction chromatography (HILIC) with fluorescence detection was successfully used to determine total CF content in multiple product types. Single-laboratory method validation results demonstrate that the method is fit for purpose for cocoa-based matrices containing <0.8 to 500 mg/g of CF.

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.

Arginase inhibitor, Nω-hydroxy-L-norarginine, spontaneously releases biologically active NO-like molecule: Limitations for research applications.

There has been a renewed interest in the enzyme arginase for its role in various physiological and pathological processes that go beyond the urea cycle. One such role ascribed to arginase has been that of regulating nitric oxide (NO) production by a substrate (l-arginine) competition between arginase and nitric oxide synthase (NOS). Several arginase inhibitors have been developed to investigate the biological roles of arginase, of which Nω-hydroxy-l-norarginine (nor-NOHA) is commercially available and is used widely from cell culture models to clinical investigations in humans. Despite the prevalence of nor-NOHA to investigate the substrate competition between arginase and NOS, little is known regarding interferences that nor-NOHA could have on common methods to assess NO production. Therefore, we investigated if nor-NOHA has unintended consequences on common NO assessment methods. We show that nor-NOHA spontaneously releases biologically active NO-like molecule in cell culture media by reacting with riboflavin. This NO-like molecule is indistinguishable from an NO donor (NOR-3) using common methods to assess NO. Besides riboflavin, nor-NOHA spontaneously reacts with H2O2 to diminish H2O2 content and produce NO-like molecule in the process. Our investigation provides detailed evidence on unintended artefacts related to nor-NOHA that can limit its use in cell culture, as well as some ex vivo and in vivo models. Future studies on arginase should take into consideration the limitations presented here when using nor-NOHA as a research tool, not only in investigations related to arginase and NOS competition, but also for investigating other biological roles of arginase.

Reliable, accessible and transferable method for the quantification of flavanols and procyanidins in foodstuffs and dietary supplements.

Flavanols and procyanidins are plant-derived bioactives that are receiving increasing attention because of their potential health benefits. Analytical tools that can accurately identify and reproducibly quantify these bioactives are critical to researchers for test material characterization, as well as to the food industry and regulators, notably for product labeling. However, the chemical complexity of procyanidins, and the absence of analytical standards have prevented the development of methods that could serve the needs of these different sectors. This report describes the development and validation of a reliable, accessible and transferable method for the quantification of flavanol monomers and procyanidins in cocoa-derived dietary supplements and foodstuffs. To accomplish this, flavanols and procyandins from cocoa, one of the most studied dietary sources of these compounds, were used as a model system. To overcome limitations related to the absence of analytical standards, a cocoa extract was thoroughly characterized for use as a calibrant. It was then used in the development and validation of a method based on reliable and accessible instrumentation, namely HPLC coupled with fluorescence detection. The resulting method permitted the quantification of flavanols and procyanidins in amounts ranging from 2 to 500 mg g-1, with high precision (%RSD 0.2 to 1.9%) and accuracy (100.7 to 102.9%). The method was successfully applied to assess the flavanol and procyanidin content of different cocoa-based commercial products. Furthermore, the high precision of the methods showed the feasibility of using principal component analysis of flavanol and procyanidin profiles to discriminate cocoa-derived products by origin and manufacturing processes. A feature that offers advantages in monitoring product authenticity/adulteration. Overall, these findings support the application of this method for the routine analysis of cocoa flavanols and procyandins.

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.