Unambiguous Characterization of Commercial Natural (Dihydro)phenanthrene Compounds Is Vital in the Discovery of AMPK Activators.

These days, easy access to commercially available (poly)phenolic compounds has expanded the scope of potential research beyond the field of chemistry, particularly in the area of their bioactivity. However, the quality of these compounds is often overlooked or not even considered. This issue is illustrated in this study through the example of (dihydro)phenanthrenes, a group of natural products present in yams, as AMP-activated protein kinase (AMPK) activators. A study conducted in our group on a series of compounds, fully characterized using a combination of chemical synthesis, NMR and MS techniques, provided evidence that the conclusions of a previous study were erroneous, likely due to the use of a misidentified commercial compound by its supplier. Furthermore, we demonstrated that additional representatives of the (dihydro)phenanthrene phytochemical classes were able to directly activate AMPK, avoiding the risk of misinterpretation of results based on analysis of a single compound alone.

Natural (dihydro)phenanthrene plant compounds are direct activators of AMPK through its allosteric drug and metabolite-binding site.

AMP-activated protein kinase (AMPK) is a central energy sensor that coordinates the response to energy challenges to maintain cellular ATP levels. AMPK is a potential therapeutic target for treating metabolic disorders, and several direct synthetic activators of AMPK have been developed that show promise in preclinical models of type 2 diabetes. These compounds have been shown to regulate AMPK through binding to a novel allosteric drug and metabolite (ADaM)-binding site on AMPK, and it is possible that other molecules might similarly bind this site. Here, we performed a high-throughput screen with natural plant compounds to identify such direct allosteric activators of AMPK. We identified a natural plant dihydrophenathrene, Lusianthridin, which allosterically activates and protects AMPK from dephosphorylation by binding to the ADaM site. Similar to other ADaM site activators, Lusianthridin showed preferential activation of AMPKß1-containing complexes in intact cells and was unable to activate an AMPKß1 S108A mutant. Lusianthridin dose-dependently increased phosphorylation of acetyl-CoA carboxylase in mouse primary hepatocytes, which led to a corresponding decrease in de novo lipogenesis. This ability of Lusianthridin to inhibit lipogenesis was impaired in hepatocytes from ß1 S108A knock-in mice and mice bearing a mutation at the AMPK phosphorylation site of acetyl-CoA carboxylase 1/2. Finally, we show that activation of AMPK by natural compounds extends to several analogs of Lusianthridin and the related chemical series, phenanthrenes. The emergence of natural plant compounds that regulate AMPK through the ADaM site raises the distinct possibility that other natural compounds share a common mechanism of regulation.

Network medicine framework shows that proximity of polyphenol targets and disease proteins predicts therapeutic effects of polyphenols.

Polyphenols, natural products present in plant-based foods, play a protective role against several complex diseases through their antioxidant activity and by diverse molecular mechanisms. Here we develop a network medicine framework to uncover mechanisms for the effects of polyphenols on health by considering the molecular interactions between polyphenol protein targets and proteins associated with diseases. We find that the protein targets of polyphenols cluster in specific neighbourhoods of the human interactome, whose network proximity to disease proteins is predictive of the molecule's known therapeutic effects. The methodology recovers known associations, such as the effect of epigallocatechin-3-O-gallate on type 2 diabetes, and predicts that rosmarinic acid has a direct impact on platelet function, representing a novel mechanism through which it could affect cardiovascular health. We experimentally confirm that rosmarinic acid inhibits platelet aggregation and α-granule secretion through inhibition of protein tyrosine phosphorylation, offering direct support for the predicted molecular mechanism. Our framework represents a starting point for mechanistic interpretation of the health effects underlying food-related compounds, allowing us to integrate into a predictive framework knowledge on food metabolism, bioavailability and drug interaction.

Journey in the Polyphenol Research World with Ragai Ibrahim.

Dr. Ragai K. Ibrahim, Professor Emeritus at Concordia University, Montréal, Canada, passed away on the November 19, 2017 at the age of 88 years. Dr. Ibrahim dedicated his entire professional life to polyphenols and spent most of his academic career (1967-1997) at the Department of Biology of Concordia University in Montréal. He has been an active member of the Groupe Polyphénols since the beginning. This paper is a tribute to Dr. Ibrahim from some of his former students. An overview of the evolution of polyphenol research since the late 1950s and the outstanding contribution that Dr. Ibrahim had to this topic is given. The input of Dr. Ibrahim's research to the enzymology and genetics of polyphenol biosynthesis is discussed. Furthermore, the links between Dr. Ibrahim's work and some aspects of modern studies on the health benefits of polyphenols are presented.

The plant product quinic acid activates Ca2+ -dependent mitochondrial function and promotes insulin secretion from pancreatic beta cells.

BACKGROUND AND PURPOSE: Quinic acid (QA) is an abundant natural compound from plant sources which may improve metabolic health. However, little attention has been paid to its effects on pancreatic beta-cell functions, which contribute to the control of metabolic health by lowering blood glucose. Strategies targeting beta-cell signal transduction are a new approach for diabetes treatment. This study investigated the efficacy of QA to stimulate beta-cell function by targeting the basic molecular machinery of metabolism-secretion coupling. EXPERIMENTAL APPROACH: We measured bioenergetic parameters and insulin exocytosis in a model of insulin-secreting beta-cells (INS-1E), together with Ca2+ homeostasis, using genetically encoded sensors, targeted to different subcellular compartments. Islets from mice chronically infused with QA were also assessed. KEY RESULTS: QA triggered transient cytosolic Ca2+ increases in insulin-secreting cells by mobilizing Ca2+ from intracellular stores, such as endoplasmic reticulum. Following glucose stimulation, QA increased glucose-induced mitochondrial Ca2+ transients. We also observed a QA-induced rise of the NAD(P)H/NAD(P)+ ratio, augmented ATP synthase-dependent respiration, and enhanced glucose-stimulated insulin secretion. QA promoted beta-cell function in vivo as islets from mice infused with QA displayed improved glucose-induced insulin secretion. A diet containing QA improved glucose tolerance in mice. CONCLUSIONS AND IMPLICATIONS: QA modulated intracellular Ca2+ homeostasis, enhancing glucose-stimulated insulin secretion in both INS-1E cells and mouse islets. By increasing mitochondrial Ca2+ , QA activated the coordinated stimulation of oxidative metabolism, mitochondrial ATP synthase-dependent respiration, and therefore insulin secretion. Bioactive agents raising mitochondrial Ca2+ in pancreatic beta-cells could be used to treat diabetes.

In Vitro Gut Metabolism of [U-13 C]-Quinic Acid, The Other Hydrolysis Product of Chlorogenic Acid.

SCOPE: Quinic acid in its free form is broadly abundant in plants, and can accumulate in copious amounts in coffee, tea, and certain fruits. However, it has been mostly studied as chlorogenic acid, an ester of caffeic and quinic acids. When chlorogenic acid reaches the colon, it is hydrolyzed by microbial esterases releasing caffeic and quinic acids. While biotransformation of chlorogenic and caffeic acids have been elucidated by in vitro and in vivo studies, the gut metabolism of quinic acid has been so far overlooked. METHODS AND RESULTS: [U-13 C]-Quinic acid is submitted to a colonic model using human fecal microbiota for assessing its metabolic fate. The metabolite profiles formed along microbial biotransformation are monitored by a combined metabolomics approach, using both 2D GC- and ultra-HPLC-MS. Six metabolic intermediates are identified by incorporation of isotopic label. CONCLUSION: Two parallel degradation pathways could be proposed: (1) an oxidative route, leading to aromatization and accumulation of protocatechuic acid, and a (2) reductive route, including dehydroxylation to cyclohexane carboxylic acid. Elucidating the biotransformation of food bioactives by the gut microbiota is of relevance for understanding nutrition, interindividual variability and potential effects on human metabolism.

Natural compounds analysis using liquid and supercritical fluid chromatography hyphenated to mass spectrometry: Evaluation of a new design of atmospheric pressure ionization source.

The MS hyphenation performance of UniSpray®, a new design of atmospheric pressure ionization source was evaluated in both SFC and RPLC modes. Sensitivity, stability, versatility and matrix effects offered by the UniSpray were assessed in positive and negative ionization modes and systematically compared to an electrospray source (ESI) using 120 natural compounds covering an extended chemical space. In a first instance, a multivariate approach was used to screen and optimize the UniSpray source settings to maximize detection sensitivity. The position of the source capillary against the fixed charged rod was highlighted as the major parameter affecting the detection sensitivity. The sensitivity improvement in Unispray vs. ESI strongly depends on the compounds chemical class and the chromatographic mode. For a few compounds (i.e. anabasine, theanine, caproic acid, fumaric acid and protopanaxatriol), up to a 10-fold increase in sensitivity was noticed with UniSpray. The signal stability over multiple injections was also found to be equivalent between both sources with RSD values on peak intensity lower than 14% on >100 injections, in both chromatographic modes. On complex plant extract, the matrix effects occurring from the secondary metabolites were also found to be comparable between ESI and UniSpray, at least in the positive ionization mode. However, a systematic decrease of MS signal intensity was observed in SFC mode when compounds were ionized using UniSpray in the negative ion mode.

Role of sulfotransferases in resveratrol metabolism in human adipocytes.

SCOPE: Polyphenols such as resveratrol received interest for their wide-ranging biological benefits, including anti-obesity potential, mimicking effects of caloric restriction with reduced body fat and increased energy expenditure. However, resveratrol is rapidly metabolized, and it is not completely understood which form of resveratrol is responsible for the effects observed within target cells such as adipocytes. Also the role of metabolizing enzymes has not been investigated before. METHODS AND RESULTS: Resveratrol metabolism was evaluated in human adipocytes by UHPLC-MS at low physiological doses. Resveratrol was found to rapidly metabolize into its sulfated form, while resveratrol glucuronides were undetectable. Only resveratrol, but not its sulfated nor glucuronidated forms had an antilipolytic effect on adipocytes. The metabolizing enzyme responsible for sulfation of polyphenols is SULT1A1, and was found to be upregulated upon adipocyte differentiation. Knocking down SULT1A1 in adipocytes led to decreased resveratrol sulfate and increased resveratrol intra- and extracellularly. This lower SULT1A1 activity resulted in an increased antilipolytic effect of resveratrol on adipocytes, as demonstrated by lower glycerol accumulation, which could be attributed to lower activity of the lipolytic protein, perilipin. CONCLUSION: Sulfotransferase activity modulates metabolism of resveratrol in adipocytes with potential consequences on bioavailability and thus metabolic action of this polyphenol.

Optimized selection of liquid chromatography conditions for wide range analysis of natural compounds.

Plant secondary metabolites are an almost unlimited reservoir of potential bioactive compounds. In view of the wide chemical space covered by natural compounds, their comprehensive analysis requires multiple and complementary approaches. In this study, numerous chromatographic conditions were tested for the analysis of a set of 120 representative natural compounds covering a wide polarity range (18 log P units). The experiments were performed on 59 different conditions involving 29 RPLC and HILIC dedicated stationary phases, as well as more exotic mixed mode columns. The best RPLC and HILIC conditions were determined using Derringer's desirability functions, based on various criteria (i.e. retention, peak shape, distribution of compounds during the gradient…). After this first selection, only the most promising conditions were kept (19 in RPLC and 11 in HILIC). The selectivity complementarity among each chromatographic mode was assessed by principal component analysis (PCA) and hierarchical cluster analysis (HCA). In RPLC, a pentabromobenzyl (PBrBz) stationary phase was identified as particularly versatile and could constitute an elegant first intention screening column. Two additional conditions allowed to extend the range of natural compounds space that can be analyzed, while offering better selectivity for basic analytes (hybrid silica graft with C8 moiety operated at pH 9 (Hyb C8)) and acidic compounds (positively charged hybrid silica graft with pentafluorophenyl moiety (Hyb+ PFPh). Although less generic in terms of amenable compounds, an ion exchange/RP mixed mode stationary phase (MM TriP1) offered notably enhanced retention of more polar analytes under RPLC conditions. With these four conditions, 89% of the natural substances were detected by LC-MS with acceptable retentions and peak shapes. In HILIC, four acceptable and complementary conditions were also highlighted. Both Syncro-Z (zwitterionic HILIC phase) and Diol columns were found to offer balanced retention and selectivity for most of the polar compounds (log DpH3<1.0). These two columns could be advantageously complemented by hybrid Amide column operated at pH 3 and Amino stationary phase at pH 5, to further enhance both retention and selectivity of polar basic and acidic species, respectively.

A novel ATP-synthase-independent mechanism coupling mitochondrial activation to exocytosis in insulin-secreting cells.

Pancreatic ß-cells sense glucose, promoting insulin secretion. Glucose sensing requires the sequential stimulation of glycolysis, mitochondrial metabolism and Ca2+ entry. To elucidate how mitochondrial activation in ß-cells contributes to insulin secretion, we compared the effects of glucose and the mitochondrial substrate methylsuccinate in the INS-1E insulin-secreting cell line at the respective concentrations at which they maximally activate mitochondrial respiration. Both substrates induced insulin secretion with distinct respiratory profiles, mitochondrial hyperpolarization, NADH production and ATP-to-ADP ratios. In contrast to glucose, methylsuccinate failed to induce large [Ca2+] rises and exocytosis proceeded largely independently of mitochondrial ATP synthesis. Both glucose- and methylsuccinate-induced secretion was blocked by diazoxide, indicating that Ca2+ is required for exocytosis. Dynamic assessment of the redox state of mitochondrial thiols revealed a less marked reduction in response to methylsuccinate than with glucose. Our results demonstrate that insulin exocytosis can be promoted by two distinct mechanisms one of which is dependent on mitochondrial ATP synthesis and large Ca2+ transients, and one of which is independent of mitochondrial ATP synthesis and relies on small Ca2+ signals. We propose that the combined effects of Ca2+ and redox reactions can trigger insulin secretion by these two mechanisms.

Standardized LC×LC-ELSD Fractionation Procedure for the Identification of Minor Bioactives via the Enzymatic Screening of Natural Extracts.

To identify natural bioactive compounds from complex mixtures such as plant extracts, efficient fractionation for biological screening is mandatory. In this context, a fully automated workflow based on two-dimensional liquid chromatography (2D-LC × LC) was developed, allowing for the production of hundreds of semipure fractions per extract. Moreover, the ELSD response was used for online sample weight estimation and automated concentration normalization for subsequent bioassays. To evaluate the efficiency of this protocol, an enzymatic assay was developed using AMP-activated protein kinase (AMPK). The activation of AMPK by nonactive extracts spiked with biochanin A, a known AMPK activator, was enhanced greatly when the fractionation workflow was applied compared to screening crude spiked extracts. The performance of the workflow was further evaluated on a red clover (Trifolium pratense) extract, which is a natural source of biochanin A. In this case, while the crude extract or 1D chromatography fractions failed to activate AMPK, semipure fractions containing biochanin A were readily localized when produced by the 2D-LC×LC-ELSD workflow. The automated fractionation methodology presented demonstrated high efficiency for the detection of bioactive compounds at low abundance in plant extracts for high-throughput screening. This procedure can be used routinely to populate natural product libraries for biological screening.

Ultra-high performance supercritical fluid chromatography coupled with quadrupole-time-of-flight mass spectrometry as a performing tool for bioactive analysis.

Secondary metabolites are an almost unlimited reservoir of potential bioactive compounds. In view of the wide chemical space covered by natural compounds, their comprehensive analysis requires multiple cutting-edge approaches. This study evaluates the applicability of ultra-high performance supercritical fluid chromatography coupled to quadrupole-time-of-flight mass spectrometry (UHPSFC-QqToF-MS) as an analytical strategy for plant metabolites profiling. Versatility of this analytical platform was first assessed using 120 highly diverse natural compounds (according to lipophilicity, hydrogen bond capability, acid-base properties, molecular mass and chemical structure) that were screened on a set of 15 rationally chosen stationary phase chemistries. UHPSFC-QqToF-MS provides a suitable analytical solution for 88% of the tested compounds. Three stationary phases (Diol, not endcapped C18 and 2-EP) were highlighted as particularly polyvalent, since they allow suitable elution of 101 out of 120 natural compounds. The systematic evaluation of retention and selectivity of natural compounds further underlined the suitability of these three columns for the separation of natural compounds. This reduced set of key stationary phases constitutes a basis for untargeted scouting analysis and method development. Even if they were less versatile, stationary phases such as endcapped T3C18, polar P-PFP, were nevertheless found to provide extended selectivity for specific natural molecules sub-classes. Finally, the identified polyvalent conditions were successfully applied for the analysis of complex polar and non-polar plant extracts. These first experimental hits demonstrate the full applicability and potential of UHPSFC-QqToF-MS for plant metabolite profiling.

Systems biology approaches for inflammatory bowel disease: emphasis on gut microbial metabolism.

Although the prevalence of main idiopathic forms of inflammatory bowel disease (IBD) has risen considerably over the last decades, their clinical features do not allow accurate prediction of prognosis, likelihood of disease progression, or response to specific therapy. Through a better understanding of the molecular pathways involved in IBD and the promise of more targeted therapies, the personalized approach to the management of IBD shows potential. To achieve this, there remains a significant need to better understand the disease process at cellular and molecular levels for any given individual with IBD. The complexity of biological functional networks behind the etiology of IBD highlights the need for their comprehensive analysis. In this, omics technologies can generate a systemic view of IBD pathogenesis on which to base novel, multiple pathway-integrated therapies. Omics sciences have just started to contribute here by generating gene, protein expression, metabolite data at global level and large scale, and more recently by offering new opportunities to explore gut functional ecology. In particular, there is much expectation regarding the putative role of the gut microbiome in IBD. No doubt it will provide additional insights and lead to the development of alternative, hopefully better, diagnostic, prognostic, and monitoring tools in the management of IBD. This review discusses perspectives of relevance to clinical translation with emphasis on gut microbial metabolic activities.

Mechanism of action of compound-13: an α1-selective small molecule activator of AMPK.

AMPK is a sensor of cellular energy status and a promising target for drugs aimed at metabolic disorders. We have studied the selectivity and mechanism of a recently described activator, C2, and its cell-permeable prodrug, C13. C2 was a potent allosteric activator of α1-complexes that, like AMP, also protected against Thr172 dephosphorylation. Compared with AMP, C2 caused only partial allosteric activation of α2-complexes and failed to protect them against dephosphorylation. We show that both effects could be fully restored by exchanging part of the linker between the autoinhibitory and C-terminal domains in α2, containing the equivalent region from α1 thought to interact with AMP bound in site 3 of the γ subunit. Consistent with our results in cell-free assays, C13 potently inhibited lipid synthesis in hepatocytes from wild-type and was largely ineffective in AMPK-knockout hepatocytes; its effects were more severely affected by knockout of α1 than of α2, ß1, or ß2.

A commentary on the role of molecular technology and automation in clinical diagnostics.

Historically, the identification of bacterial or yeast isolates has been based on phenotypic characteristics such as growth on defined media, colony morphology, Gram stain, and various biochemical reactions, with significant delay in diagnosis. Clinical microbiology as a medical specialty has embraced advances in molecular technology for rapid species identification with broad-range 16S rDNA polymerase chain reaction (PCR) and matrix-assisted laser desorption and/or ionization time of flight (MALDI-TOF) mass spectrometry demonstrated as accurate, rapid, and cost-effective methods for the identification of most, but not all, bacteria and yeasts. Protracted conventional incubation times previously necessary to identify certain species have been mitigated, affording patients quicker diagnosis with associated reduction in exposure to empiric broad-spectrum antimicrobial therapy and shortened hospital stay. This short commentary details such molecular advances and their implications in the clinical microbiology setting.

Dose-response plasma appearance of coffee chlorogenic and phenolic acids in adults.

SCOPE: Coffee contains phenolic compounds, mainly chlorogenic acids (CGAs). Even though coffee intake has been associated with some health benefits in epidemiological studies, the bioavailability of coffee phenolics is not fully understood. OBJECTIVE AND STUDY DESIGN: We performed a dose-response study measuring plasma bioavailability of phenolics after drinking three increasing, but still nutritionally relevant doses of instant pure soluble coffee. The study design was a one treatment (coffee) three-dose randomized cross-over design, with a washout period of 2 wks between visits. RESULTS: CGAs, phenolic acids, and late-appearing metabolites all increased with increasing ingested dose. Hence, the sum of area under the curve was significantly higher for the medium to low dose, and high to medium dose, by 2.23- and 2.38-fold, respectively. CGAs were not well absorbed in their intact form, regardless of the dose. CGA and phenolic acids appeared rapidly in plasma, indicating an early absorption in the gastrointestinal tract. Late-appearing metabolites were the most abundant, regardless of the dose. CONCLUSION: This study confirmed previous findings about coffee bioavailability but also showed that coffee phenolics appear in a positive dose-response manner in plasma when drank at nutritionally relevant doses.

Identification of cholesteryl ester of ferulic acid in human plasma by mass spectrometry.

Epidemiological data suggests that regular consumption polyphenol rich foods and beverages is associated with a reduced risk of certain pathological conditions. While the in vivo "per se" antioxidant benefit of polyphenols still has not been clearly demonstrated, it has been suggested that phenolic acids can be incorporated into low-density lipoproteins (LDL). In the present study, we hypothesized that esterification of phenolic acids - such as ferulic acid - with lipophilic substances such as cholesterol can occur in vivo. To prove this hypothesis, we have synthesized pure cholesteryl-ferulate standard and used gas- and liquid chromatography coupled with mass spectrometry to confirm the presence of endogenous form in human plasma. The detection and identification of cholesteryl ferulate was based on: (1) matching gas- and liquid chromatographic retention time with the reference standard; (2) accurate mass of the molecular ion; (3) matching electron ionization mass spectrum and (4) matching electrospray product ion spectrum. The identified cholesteryl ferulate demonstrated an in vitro antioxidant capacity in various assays. The present study confirmed that phenolic acid can be found in human plasma as lipophilic conjugates which exert antioxidant capacity. These molecules can potentially be involved in the protection of lipoproteins against oxidative damages.

Dose-dependent absorption of chlorogenic acids in the small intestine assessed by coffee consumption in ileostomists.

SCOPE: Until now, the question of how the ingested doses of chlorogenic acids (CGA) from coffee influence their absorption and metabolism remains unresolved. To assess absorption in the small intestine, we performed a dose-response study with a randomized, double-blinded, crossover design with ileostomist subjects. METHODS AND RESULTS: After a polyphenol-free diet, the volunteers consumed, on three separate occasions, coffee with different total CGA contents (high 4525 µmol; medium 2219 µmol; low 1053 µmol). CGA concentrations in plasma, ileal effluent, and urine were subsequently determined by HPLC-DAD-ESI-MS and -ESI-MS/MS. The results show that the consumption of higher CGA concentrations leads to a faster ileal excretion. This corresponds to a renal excretion of 8.0 ± 4.9% (high), 12.1 ± 6.7% (medium), and 14.6 ± 6.8% (low) of total CGA and metabolites. Glucuronidation of CGA became slightly greater with increasing dose. After enzyme treatment, the area under the curve (AUC)(0-8h) for CGA metabolites in plasma was 4412 ± 751 nM × h(0-8) (-1) (high), 2394 ± 637 nM × h(0-8) (-1) (medium), 1782 ± 731 nM × h(0-8) (-1) (low), respectively. Additionally, we were able to identify new metabolites of CGA in urine and ileal fluid. CONCLUSION: We conclude that the consumption of high CGA concentrations via coffee might influence the gastrointestinal transit time and consequently affect CGA absorption and metabolism.

Epicatechin B-ring conjugates: first enantioselective synthesis and evidence for their occurrence in human biological fluids.

Herein, the first enantioselective total synthesis of a number of biologically relevant (-)-epicatechin conjugates is described. The success of this synthesis relied on (i) optimized conditions for the stereospecific cyclization step leading to the catechin C ring; on (ii) efficient conjugation reactions; and on (iii) optimized deprotection sequences. These standard compounds have been subsequently used to elucidate for the first time the pattern of (-)-epicatechin conjugates present in four different human biological fluids following (-)-epicatechin absorption.

Elucidation of (-)-epicatechin metabolites after ingestion of chocolate by healthy humans.

After absorption in the gastrointestinal tract, (-)-epicatechin is extensively transformed into various conjugated metabolites. These metabolites, chemically different from the aglycone forms found in foods, are the compounds that reach the circulatory system and the target organs. Therefore, it is imperative to identify and quantify these circulating metabolites to investigate their roles in the biological effects associated with (-)-epicatechin intake. Using authentic synthetic standards of (-)-epicatechin sulfates, glucuronides, and O-methyl sulfates, a novel LC-MS/MS-MRM analytical methodology to quantify (-)-epicatechin metabolites in biological matrices was developed and validated. The optimized method was subsequently applied to the analysis of plasma and urine metabolites after consumption of dark chocolate, an (-)-epicatechin-rich food, by humans. (-)-Epicatechin-3'-ß-d-glucuronide (C(max) 290 ± 49 nM), (-)-epicatechin 3'-sulfate (C(max) 233 ± 60 nM), and 3'-O-methyl epicatechin sulfates substituted in the 4', 5, and 7 positions were the most relevant (-)-epicatechin metabolites in plasma. When plasmatic metabolites were divided into their substituent groups, it was revealed that (-)-epicatechin glucuronides, sulfates, and O-methyl sulfates represented 33 ± 4, 28 ± 5, and 33 ± 4% of total metabolites (AUC(0-24)(h)), respectively, after dark chocolate consumption. Similar metabolites were found in urine samples collected over 24h. The total urine excretion of (-)-epicatechin was 20 ± 2% of the amount ingested. In conclusion, we describe the entire metabolite profile and its degree of elimination after administration of (-)-epicatechin-containing food. These results will help us understand more precisely the mechanisms and the main metabolites involved in the beneficial physiological effects of flavanols.