Over the last decade, a growing body of interventional and epidemiological studies has furnished new evidence on the beneficial properties of dietary polyphenols in the reduction of the risk of cardiovascular disease (CVD). However, further research is required on the discovery and validation of new polyphenol intake biomarkers and their effect on the promotion of cardiovascular health. To successfully face the challenge of biomarker discovery, the application of emerging “omics” technologies and hypothesis-generating approaches are very encouraging. Metabolomics is a particularly promising strategy for obtaining a comprehensive overview of the low-molecular-weight metabolites present in a biological system at a given moment, and to explore unknown metabolic pathways impacted on diet. Despite being the youngest of the “omics” technologies, it already counts for a series of successful applications in the identification of potential biomarkers related to the prediction, early diagnosis and prognosis of CVD. This chapter will review the state of the art in the application of the metabolomics approach to polyphenol and CVD biomarker discovery, and their implications in cardiovascular risk assessment.

The study of the bioavailability of active compounds in functional foods, such as polyphenol‐rich beverages, is required before making nutritional claims. In this work, we aimed to study the urinary excretion of resveratrol (RV), taking into consideration its gut and microbial metabolites after consumption of a functional beverage (FB), applying a ultra performance liquid chromatography (UPLC)‐MS/MS methodology. A randomized, crossover, placebo‐controlled, double‐blind intervention study was performed with 26 volunteers, who consumed 187 mL of a control placebo or a FB in an acute study, and twice a day during 15 days for a chronic consumption study. The whole profile of 21 RV metabolites increased after acute and chronic consumption of the FB with respect to the control‐placebo beverage and to the baseline. Urinary excretion of RV and piceid phase II metabolites was similar after both consumption periods, but a later formation of microbial metabolites required urine sampling of up to 24 h after the consumption of the FB. In addition, the intervariability has been evaluated. This study allows the knowledge of the RV metabolites that reach target tissues where biological activity would be achieved in order to elucidate the beneficial effects of this grape extract FB.

The health-promoting effects attributable to dietary phenolic compounds strongly depend on their bioaccesibility from the food matrix and their consequent bioavailability. We carried out a pilot randomized controlled cross-over study to evaluate the effect of addition of an oil matrix during tomato sauce processing, on the bioavailability of tomato phenolics. Healthy subjects consumed a single dose of tomato sauce elaborated without oil (OO-F) and with the addition of 5% virgin olive oil (VOO-E) or refined olive oil (ROO-E). Plasma and urine samples were subjected to solid-phase extraction, followed by HPLC–MS/MS analysis. Six phenolic compounds, three aglycones (naringenin, ferulic and caffeic acids) and their corresponding glucuronide metabolites, were identified and quantified in urine after the ingestion of the tomato sauces. Two of the six phenolic urinary metabolites were also quantified in plasma samples. Only after ingestion of the oil-enriched tomato sauces, did the glucuronide metabolites of naringenin show a bi-phasic profile of absorption in plasma, suggesting that the lipid matrix added to the sauce may stimulate the occurrence of re-absorption events by enterohepatic circulation, potentially enhancing the apparent plasma half-life of the flavanone prior to excretion. The interindividual response variability observed underlies the need for further large-scale investigations.

Resveratrol exerts a variety of biological and pharmacological activities, which are observed despite its extremely low bioavailability and rapid clearance from the circulation due to extensive sulfation and glucuronidation in the intestine and liver. In order to more accurately quantify all known resveratrol metabolites, a sensitive and optimized analytical assay was developed and validated by pure standards. Methodology improvements aimed to the chromatographic detection of disulfates and sulfoglucuronides, improving resolution of sulfates, by using a buffered solution, with recovery values of resveratrol and its metabolites, even of sulfates, of 99%. The adapted methodology was then applied to a clinical study with high cardiovascular risk subjects, after the moderate consumption of red wine (RW) or dealcoholized red wine (DRW) for 28 days. Up to 21 resveratrol metabolites, including those formed by gut and microbial metabolism, were identified in 24-h urine samples. Interestingly, after long-term consumption of RW and DRW, resveratrol metabolite concentration significantly increased in urine with no differences between the two interventions, indicating that bioavailability and biotransformation of resveratrol is not affected by the alcoholic matrix of wine. In summary, we established a sensitive analytical assay for the quantification of a wide resveratrol metabolic profile in human urine, also regarding gut microbial-derived metabolites, which may also be applied to blood and tissue samples. The resveratrol metabolic pattern might therefore act as an excellent marker for the efficacy of resveratrol in clinical and epidemiological studies for the study of the beneficial effects of grape product consumption. In this sense, having a more precise concentration value of all the resveratrol metabolites in target tissues would finally lead to a better interpretation of the obtained results.

This study aimed to characterize resveratrol metabolite profiles in liver, skeletal muscle, and adipose tissue in rats treated for 6 weeks with 6, 30, or 60 mg of trans-resveratrol/kg body weight/d. Resveratrol metabolites were quantified by liquid chromatography-tandem mass spectrometry. The greatest number of metabolites was found in liver followed by adipose tissue. A great number of metabolites in muscle was below the limit of detection. The amounts of sulfate conjugates tended to increase when resveratrol dosage was enhanced, while the glucuronide ones increased only between 6 and 30 mg/kg/d. Microbiota metabolites were detected in higher amounts than resveratrol conjugates in liver, while the opposite occurred in adipose tissue and muscle. So, the largest amounts of resveratrol metabolites were found in liver, intermediate amounts in adipose tissue, and the lowest amounts in muscle. Sulfate conjugates, but not glucuronides, showed a dose response pattern. Microbiota metabolites were predominant in liver.

A pharmacokinetic study of the metabolic profile of resveratrol has been performed in healthy men after moderate red wine (RW) consumption. The bioavailability of resveratrol is highly influenced by several factors such as the food matrix and, therefore, this study has been compared with a pilot study in which men ingested grape extract (GE) tablets as a nutraceutical, containing similar total amounts of resveratrol than RW. Blood and urine samples were taken before and at several time points after intervention and then analyzed by SPE and LC–ESI–MS/MS. Up to 17 resveratrol and piceid derivatives were identified, including those formed by the intestinal microbiota. Resveratrol glucosides were found in plasma as intact forms and reached the lowest maximum concentrations 1h after both interventions. Higher plasma concentrations and longer times (tmax) were observed for resveratrol glucuronides due to phase II metabolism and even higher values for conjugates derived from microbiota, such as dihydroresveratrol-glucuronides. The same trend was observed for total excreted amounts in urine samples. When both treatments were compared, statistically significant differences for some metabolites were obtained, which may be due to the different composition of resveratrol and piceid in both sources. However, GE formulation seems to delay resveratrol absorption, staying longer in the gut where could be metabolized to a greater degree, since 2.1–3.6-fold higher urinary concentrations of microbial metabolites were observed after GE intervention at 12–24h urinary fraction. Therefore, supplement intake could be also a way to bring resveratrol benefits to human health.

Flavonoids are a widely distributed group of polyphenolic compounds present in an extensive range of edible plants, notably Citrus species. This article reports a rapid, optimized, and validated method for the separation and quantification of flavonoids in three Citrus fruit extracts by ultrahigh performance liquid chromatography (UHPLC) using a photodiode array detector. This new procedure allowed the simultaneous separation and quantification of 11 selected flavonoids in 5.5 min, 8.2 times faster than that by HPLC analysis. The solvent consumption for each individual analysis was also reduced almost 6.2-fold. The most abundant component in the analyzed samples was naringin (299.06-544.36 mg 100 g⁻¹), followed by rutin (116.60-256.33 mg 100 g⁻¹) and quercetin (7.78-251.49 mg 100 g⁻¹). Isoquercitrin was found in a lower proportion (60.05-81.88 mg 100 g⁻¹). The method was completely validated, providing a sensitive analysis for flavonoid detection and showing satisfactory data for all the parameters tested. This methodology is cheaper, more environmentally friendly, and easier to perform than others previously described.

BACKGROUNDFew clinical studies have focused on the alcohol-independent cardiovascular effects of the phenolic compounds of red wine (RW).OBJECTIVEWe aimed to evaluate the effects of ethanol and phenolic compounds of RW on the expression of inflammatory biomarkers related to atherosclerosis in subjects at high risk of cardiovascular disease.DESIGNSixty-seven high-risk, male volunteers were included in a randomized, crossover consumption trial. After a washout period, all subjects received RW (30 g alcohol/d), the equivalent amount of dealcoholized red wine (DRW), or gin (30 g alcohol/d) for 4 wk. Before and after each intervention period, 7 cellular and 18 serum inflammatory biomarkers were evaluated.RESULTSAlcohol increased IL-10 and decreased macrophage-derived chemokine concentrations, whereas the phenolic compounds of RW decreased serum concentrations of intercellular adhesion molecule-1, E-selectin, and IL-6 and inhibited the expression of lymphocyte function-associated antigen 1 in T lymphocytes and macrophage-1 receptor, Sialil-Lewis X, and C-C chemokine receptor type 2 expression in monocytes. Both ethanol and phenolic compounds of RW downregulated serum concentrations of CD40 antigen, CD40 ligand, IL-16, monocyte chemotactic protein-1, and vascular cell adhesion molecule-1.CONCLUSIONThe results suggest that the phenolic content of RW may modulate leukocyte adhesion molecules, whereas both ethanol and polyphenols of RW may modulate soluble inflammatory mediators in high-risk patients. The trial was registered in the International Standard Randomized Controlled Trial Number Register at http://www.isrctn.org/ as ISRCTN88720134.

Numerous health benefits have been attributed to cocoa and its derived products in the last decade including antioxidant, anti-platelet and positive effects on lipid metabolism and vascular function. Inflammation plays a key role in the initiation and progression of atherosclerosis. However, cocoa feeding trials focused on inflammation are still rare and the results yielded are controversial. Health effects derived from cocoa consumption have

Resveratrol (a natural stilbene) intake has been associated with several health-promoting activities. In recent years it has become a promising molecule, mainly for the possible development of future treatments for cardiovascular and aging-related diseases. Despite its health implications, other aspects such as dietary intake and bioavailability should be considered before making nutritional recommendations or proposing treatment guidelines. In this context, this review will attempt to provide evidence of these aspects and will also propose possible approaches for the future towards gaining an understanding of the links between resveratrol intake and health.

Health effects of cocoa flavonols depend on their bioavailability, which is strongly influenced by the food matrix and the degree of flavanol polymerization. The effect of milk on the bioavailability of cocoa flavanoids considering phase II metabolites of epicatechin has been the subject of considerable debate. This work studies the effect of milk at the colonic microbial metabolism level of the nonabsorbed flavanol fraction that reaches the colon and is metabolized by the colonic microbiota into various phenolic acids. Twenty-one human volunteers followed a diet low in polyphenols for at least 48 h before taking, in a random order, 40 g of cocoa powder dissolved either in 250 mL of whole milk or in 250 mL of water. Urine samples were collected before the intake and during three different periods (0-6, 6-12, and 12-24 h). Phenolic acids were analyzed by LC-MS/MS after solid-phase extraction. Of the 15 metabolites assessed, the excretion of 9 phenolic acids was affected by the intake of milk. The urinary concentration of 3,4-dihydroxyphenylacetic, protocatechuic, 4-hydroxybenzoic, 4-hydroxyhippuric, hippuric, caffeic, and ferulic acids diminished after the intake of cocoa with milk, whereas urinary concentrations of vanillic and phenylacetic acids increased. In conclusion, milk partially affects the formation of microbial phenolic acids derived from the colonic degradation of procyanidins and other compounds present in cocoa powder.

Beer is one of the most commonly consumed undistilled alcoholic beverages in many countries. In recent studies, the stilbenes resveratrol and piceid have been found in some hop varieties which are used in the production of beer. Therefore, they could be transferred to beer. The aim of the present work was to validate a method to study the potential content of trans- and cis-resveratrol and piceid in 110 commercial beers from around the world. The resveratrol and piceid contents of 110 beers were analyzed by liquid chromatography–tandem mass spectrometry (LC–MS/MS) after a solid-phase extraction (SPE) using optimized and validated procedures for the beer matrix. The beer matrix effect was also studied. Stilbenes were found in quantifiable amounts in 92 beers, while concentrations below the limit of quantification (LOQ) were found in 18 beers. Resveratrol was found in the range of 1.34–77.0μg/L in 79% of the beers analyzed, and piceid was found in the range of 1.80–27.3μg/L in only 33% of them. The mean of total resveratrol in all the beers was 14.7±20.5μg/L. The content of resveratrol has been compared with other resveratrol containing foods. A serving of beer contains similar amounts of stilbenes as berries, less than chocolate and grape products but more than pistachios, peanuts or tomatoes. Overall, beer is one of the products with the lowest levels of total resveratrol (μg/L), and despite its high consumption it should not be considered as a representative source of resveratrol.