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Although LC-MS untargeted metabolomics continues to expand into exiting research domains, methodological issues have not been solved yet by the definition of unbiased, standardized and globally accepted analytical protocols. In the present study, the response of the plasma metabolome coverage to specific methodological choices of the sample preparation (two SPE technologies, three sample-to-solvent dilution ratios) and the LC-ESI-MS data acquisition steps of the metabolomics workflow (four RP columns, four elution solvent combinations, two solvent quality grades, postcolumn modification of the mobile phase) was investigated in a pragmatic and decision tree-like performance evaluation strategy. Quality control samples, reference plasma and human plasma from a real nutrimetabolomic study were used for intermethod comparisons. Uni- and multivariate data analysis approaches were independently applied. The highest method performance was obtained by combining the plasma hybrid extraction with the highest solvent proportion during sample preparation, the use of a RP column compatible with 100% aqueous polar phase (Atlantis T3), and the ESI enhancement by using UHPLC-MS purity grade methanol as both organic phase and postcolumn modifier. Results led to the following considerations: submit plasma samples to hybrid extraction for removal of interfering components to minimize the major sample-dependent matrix effects; avoid solvent evaporation following sample extraction if loss in detection and peak shape distortion of early eluting metabolites are not noticed; opt for a RP column for superior retention of highly polar species when analysis fractionation is not feasible; use ultrahigh quality grade solvents and "vintage" analytical tricks such as postcolumn organic enrichment of the mobile phase to enhance ESI efficiency. The final proposed protocol offers an example of how novel and old-fashioned analytical solutions may fruitfully cohabit in untargeted metabolomics protocols.

Scope: The aim of the current study was to apply an untargeted metabolomics strategy to characterize a model of cocoa intake biomarkers in a free-living population., Methods and Results: An untargeted HPLC-q-ToF-MS based metabolomics approach was applied to human urine from 32 consumers of cocoa or derived products (CC) and 32 matched control subjects with no consumption of cocoa products (NC). The multivariate statistical analysis (OSC-PLS-DA) showed clear differences between CC and NC groups. The discriminant biomarkers identified were mainly related to the metabolic pathways of theobromine and polyphenols, as well as to cocoa processing. Consumption of cocoa products was also associated with reduced urinary excretions of methylglutarylcarnitine, which could be related to effects of cocoa exposure on insulin resistance. To improve the prediction of cocoa consumption, a combined urinary metabolite model was constructed. ROC curves were performed to evaluate the model and individual metabolites. The AUC values (95% CI) for the model were 95.7% (89.8-100%) and 92.6% (81.9-100%) in training and validation sets, respectively, whereas the AUCs for individual metabolites were <90%., Conclusions: The metabolic signature of cocoa consumption in free-living subjects reveals that combining different metabolites as biomarker models improves prediction of dietary exposure to cocoa., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The Mediterranean diet (MD) is considered a dietary pattern with beneficial effects on human health. The aim of this study was to assess the effect of an MD on urinary metabolome by comparing subjects at 1 and 3 years of follow-up, after an MD supplemented with either extra-virgin olive oil (MD + EVOO) or nuts (MD + Nuts), to those on advice to follow a control low-fat diet (LFD). Ninety-eight nondiabetic volunteers were evaluated, using metabolomic approaches, corresponding to MD + EVOO (n = 41), MD + Nuts (n = 27), or LFD (n = 30) groups. The (1)H NMR urinary profiles were examined at baseline and after 1 and 3 years of follow-up. Multivariate data analysis (OSC-PLS-DA and HCA) methods were used to identify the potential biomarker discriminating groups, exhibiting a urinary metabolome separation between MD groups against baseline and LFD. Results revealed that the most prominent hallmarks concerning MD groups were related to the metabolism of carbohydrates (3-hydroxybutyrate, citrate, and cis-aconitate), creatine, creatinine, amino acids (proline, N-acetylglutamine, glycine, branched-chain amino acids, and derived metabolites), lipids (oleic and suberic acids), and microbial cometabolites (phenylacetylglutamine and p-cresol). Otherwise, hippurate, trimethylamine-N-oxide, histidine and derivates (methylhistidines, carnosine, and anserine), and xanthosine were predominant after LFD. The application of NMR-based metabolomics enabled the classification of individuals regarding their dietary pattern and highlights the potential of this approach for evaluating changes in the urinary metabolome at different time points of follow-up in response to specific dietary interventions.

Unlabelled: Liquid chromatography coupled to mass spectrometry (LC/MS) has become widely used in Metabolomics. Several artefacts have been identified during the acquisition step in large LC/MS metabolomics experiments, including ion suppression, carryover or changes in the sensitivity and intensity. Several sources have been pointed out as responsible for these effects. In this context, the drift effects of the peak intensity is one of the most frequent and may even constitute the main source of variance in the data, resulting in misleading statistical results when the samples are analysed. In this article, we propose the introduction of a methodology based on a common variance analysis before the data normalization to address this issue. This methodology was tested and compared with four other methods by calculating the Dunn and Silhouette indices of the quality control classes. The results showed that our proposed methodology performed better than any of the other four methods. As far as we know, this is the first time that this kind of approach has been applied in the metabolomics context., Availability and Implementation: The source code of the methods is available as the R package intCor at http://b2slab.upc.edu/software-and-downloads/intensity-drift-correction/., Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Infectious mastitis is a common condition among lactating women, with staphylococci and streptococci being the main aetiological agents. In this context, some lactobacilli strains isolated from breast milk appear to be particularly effective for treating mastitis and, therefore, constitute an attractive alternative to antibiotherapy. A (1)H NMR-based metabolomic approach was applied to detect metabolomic differences after consuming a probiotic strain (Lactobacillus salivarius PS2) in women with mastitis. 24h urine of women with lactational mastitis was collected at baseline and after 21 days of probiotic (PB) administration. Multivariate analysis (OSC-PLS-DA and hierarchical clustering) showed metabolome differences after PB treatment. The discriminant metabolites detected at baseline were lactose, and ibuprofen and acetaminophen (two pharmacological drugs commonly used for mastitis pain), while, after PB intake, creatine and the gut microbial co-metabolites hippurate and TMAO were detected. In addition, a voluntary desertion of the pharmacological drugs ibuprofen and acetaminophen was observed after probiotic administration. The application of NMR-based metabolomics enabled the identification of the overall effects of probiotic consumption among women suffering from mastitis and highlighted the potential of this approach in evaluating the outcomes of probiotics consumption. To our knowledge, this is the first time that this approach has been applied in women with mastitis during lactation., (Copyright © 2014. Published by Elsevier Ltd.)

The health benefits associated with the consumption of polyphenol-rich foods have been studied in depth, however, the full mechanism of action remains unknown. One of the proposed mechanisms is through microbiota interaction. In the present study, we aimed to explore the relationship between changes in fecal microbiota and changes in urinary phenolic metabolites after wine interventions. Nine participants followed a randomized, crossover, controlled interventional trial. After the washout period, they received red wine, dealcoholized red wine or gin for 20 days each. Polyphenol metabolites (n > 60) in urine were identified and quantified by UPLC-MS/MS and the microbial content of fecal samples was quantified by real-time quantitative PCR. Interventions with both red wine and dealcoholized red wine increased the fecal concentration of Bifidobacterium, Enterococcus and Eggerthella lenta, compared to gin intervention and baseline. When participants were categorized in tertiles of changes in fecal bacteria, those in the highest tertile of Bifidobacteria had higher urinary concentration changes in syringic acid, p-coumaric acid, 4-hydroxybenzoic acid and homovanillic acid (all anthocyanin metabolites) than those in tertile 1 (P < 0.05, all). In addition, changes of Bifidobacteria correlated positively with changes of these metabolites (r = 0.5-0.7, P < 0.05, all). Finally, the 68.5% changes in Bifidobacteria can be predicted by syringic acid and 4-hydroxybenzoic acid changes. This study confirms the important role of polyphenols as bacterial substrates and their modulatory capacity as an important field in the research of new products with prebiotic and probiotic characteristics for the food industry.

The beneficial impact of walnuts on human health has been attributed to their unique chemical composition. In order to characterize the dietary walnut fingerprinting, spot urine samples from two sets of 195 (training) and 186 (validation) individuals were analyzed by an HPLC-q-ToF-MS untargeted metabolomics approach, selecting the most discriminating metabolites by multivariate data analysis (VIP ≥ 1.5). Stepwise logistic regression analysis was used to design a multimetabolite prediction biomarker model. The global performance of the model and each included metabolite in it was evaluated by receiver operating characteristic curves, using the area under the curve (AUC) values. Dietary exposure to walnuts was characterized by 18 metabolites, including markers of fatty acid metabolism, ellagitannin-derived microbial compounds, and intermediate metabolites of the tryptophan/serotonin pathway. The predictive model of walnut exposure included at least one compound of each class. The AUC (95% CI) for the combined biomarker model was 93.4% (90.1-96.8%) in the training set and 90.2% (85.9-94.6%) in the validation set. The AUCs for individual metabolites were ≤85%. As far as we know, this is the first study proposing a combination of biomarkers of walnut exposure in a population under free-living conditions, as considered in epidemiological studies examining associations between diet and health outcomes.

Importance: Resveratrol, a polyphenol found in grapes, red wine, chocolate, and certain berries and roots, is considered to have antioxidant, anti-inflammatory, and anticancer effects in humans and is related to longevity in some lower organisms., Objective: To determine whether resveratrol levels achieved with diet are associated with inflammation, cancer, cardiovascular disease, and mortality in humans., Design: Prospective cohort study, the Invecchiare in Chianti (InCHIANTI) Study ("Aging in the Chianti Region"), 1998 to 2009 conducted in 2 villages in the Chianti area in a population-based sample of 783 community-dwelling men and women 65 years or older., Exposures: Twenty-four-hour urinary resveratrol metabolites., Main Outcomes and Measures: Primary outcome measure was all-cause mortality. Secondary outcomes were markers of inflammation (serum C-reactive protein [CRP], interleukin [IL]-6, IL-1β, and tumor necrosis factor [TNF]) and prevalent and incident cancer and cardiovascular disease., Results: Mean (95% CI) log total urinary resveratrol metabolite concentrations were 7.08 (6.69-7.48) nmol/g of creatinine. During 9 years of follow-up, 268 (34.3%) of the participants died. From the lowest to the highest quartile of baseline total urinary resveratrol metabolites, the proportion of participants who died from all causes was 34.4%, 31.6%, 33.5%, and 37.4%, respectively (P = .67). Participants in the lowest quartile had a hazards ratio for mortality of 0.80 (95% CI, 0.54-1.17) compared with those in the highest quartile of total urinary resveratrol in a multivariable Cox proportional hazards model that adjusted for potential confounders. Resveratrol levels were not significantly associated with serum CRP, IL-6, IL-1β, TNF, prevalent or incident cardiovascular disease, or cancer., Conclusions and Relevance: In older community-dwelling adults, total urinary resveratrol metabolite concentration was not associated with inflammatory markers, cardiovascular disease, or cancer or predictive of all-cause mortality. Resveratrol levels achieved with a Western diet did not have a substantial influence on health status and mortality risk of the population in this study.

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., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The food metabolome is defined as the part of the human metabolome directly derived from the digestion and biotransformation of foods and their constituents. With >25,000 compounds known in various foods, the food metabolome is extremely complex, with a composition varying widely according to the diet. By its very nature it represents a considerable and still largely unexploited source of novel dietary biomarkers that could be used to measure dietary exposures with a high level of detail and precision. Most dietary biomarkers currently have been identified on the basis of our knowledge of food compositions by using hypothesis-driven approaches. However, the rapid development of metabolomics resulting from the development of highly sensitive modern analytic instruments, the availability of metabolite databases, and progress in (bio)informatics has made agnostic approaches more attractive as shown by the recent identification of novel biomarkers of intakes for fruit, vegetables, beverages, meats, or complex diets. Moreover, examples also show how the scrutiny of the food metabolome can lead to the discovery of bioactive molecules and dietary factors associated with diseases. However, researchers still face hurdles, which slow progress and need to be resolved to bring this emerging field of research to maturity. These limits were discussed during the First International Workshop on the Food Metabolome held in Glasgow. Key recommendations made during the workshop included more coordination of efforts; development of new databases, software tools, and chemical libraries for the food metabolome; and shared repositories of metabolomic data. Once achieved, major progress can be expected toward a better understanding of the complex interactions between diet and human health., (© 2014 American Society for Nutrition.)

Liquid chromatography-mass spectrometry (LC-MS)-based metabolomic datasets consist of different features including (de)protonated molecules, fragments, adducts, and isotopes that may show high correlation values related to a high level of collinearity. There have been described several sources of these high correlation patterns regarding metabolomic datasets. Among these sources, it should be highlighted the high level of correlation computed between features coming from the same metabolite. It is well-known that soft ionization methods (such as electrospray) produce several mass features from a particular compound (i.e., metabolite spectrum). Typically, the statistical methods used in metabolomics consider spectral peaks as variables. However, it has been reported that a high collinearity between variables might be the responsible for high uncertainty values in the predictors of a regression. In this context, this technical note proposes a new strategy based on the application of the so-called peak aggregation methods (NMF Reduction, PCA Decomposition, Maximum Peak, and Spectrum Mean) to take advantage of the variable collinearity and solve the issue of high variable collinearity. A set of real samples obtained after human nutritional intervention with placebo or polyphenol-rich beverages was used to test this methodology. The results showed that applying any peak aggregation method (especially NMF and PCA) improves the statistical prediction power of class pertinence independently of the nature of the classifier (linear PLS-DA or nonlinear SVM). Overall, the introduction of this new approach resulted in a reduction of the dimensionality of the data and, in addition, in a significant increase in the overall predictive power of the data.

Scope: Knowledge of in vivo polyphenol metabolites derived from the consumption of red wine could be key to understanding its health benefits. This work aimed to predict the wine polyphenol metabolic space in biofluids by using all available data compiled in the Phenol-Explorer database., Methods and Results: A search strategy was developed for Phenol-Explorer to obtain the widest range of metabolites related to wine consumption. A total of 97 metabolites have been described in intervention studies with wine and related products (n = 37), and after consumption of pure compounds known to be wine constituents (n = 90). These 97 metabolites, derived from host and microbial metabolism of several classes of polyphenols, were found in plasma and urine samples and some of them have demonstrated higher or lower biological activities than the parent compound in in vitro studies. The metabolites have been linked to generate, for the first time, a global pathway map of wine in vivo polyphenol metabolism., Conclusion: The retrieval of the widest range of metabolites so far described and their assembly as a metabolic pathway map could aid the identification of possible biomarkers of wine consumption and improve current understanding of the health effects of wine consumption., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Epidemiological studies have demonstrated the beneficial effect of plant-derived food intake in reducing the risk of cardiovascular disease (CVD). The potential bioactivity of cocoa and its polyphenolic components in modulating cardiovascular health is now being studied worldwide and continues to grow at a rapid pace. In fact, the high polyphenol content of cocoa is of particular interest from the nutritional and pharmacological viewpoints. Cocoa polyphenols are shown to possess a range of cardiovascular-protective properties, and can play a meaningful role through modulating different inflammatory markers involved in atherosclerosis. Accumulated evidence on related anti-inflammatory effects of cocoa polyphenols is summarized in the present review.

The regular consumption of dealcoholized red wine (DRW) has demonstrated benefits in cardiovascular risk factors. The analysis of phenolic metabolites formed in the organism, especially those that could come from microbiota metabolism, would help to understand these benefits. The aim of this study was to determine the widest urinary metabolomic fingerprinting of phenolics and microbial-derived phenolic acids (n = 61) after regular intake of DRW in men at high cardiovascular risk by UPLC-MS/MS using a targeted approach. Up to 49 metabolites, including phase II and microbial phenolic metabolites, increased after DRW consumption compared to baseline (P < 0.05). The highest percentage of increase was found for microbial metabolites from anthocyanin degradation such as syringic, p-coumaric, gallic acids and pyrogallol and from flavan-3-ols degradation such as hydroxyphenylvalerolactones and (epi)catechins. These findings provide the most complete metabolic fingerprinting after wine consumption, amplifying the spectrum of microbial derived metabolites and their potential bioactivity related with health benefits.

Polyphenols might have a role in the prevention of several chronic diseases, but evaluating total dietary polyphenol (TDP) intake from self-reported questionnaires is inaccurate and unreliable. A promising alternative is to use total urinary polyphenol (TUP) concentration as a proxy measure of intake. The current study evaluated the relationship between TUPs and TDPs and all-cause mortality during a 12-y period among older adult participants. The study population included 807 men and women aged 65 y and older from the Invecchiare in Chianti study, a population-based cohort study of older adults living in the Chianti region of Tuscany, Italy. TUP concentrations were measured at enrolment (1998-2000) using the Folin-Ciocalteau assay after a solid-phase extraction. TDPs were also estimated at baseline throughout a validated food frequency questionnaire and using our database based on USDA and Phenol-Explorer databases. We modeled associations using Kaplan-Meier survival and Cox proportional hazards models, with adjustment for potential confounders. During the 12-y follow-up, 274 participants (34%) died. At enrollment, TUP excretion adjusted for age and sex tended to be greater in participants who survived [163 ± 62 mg gallic acid equivalents (GAE)/d)] than in those who died (143 ± 63 mg GAE/d) (P = 0.07). However, no significant differences were observed for TDPs. In the multivariable Cox model, participants in the highest tertile of TUP at enrolment had a lower mortality rate than those in the lowest tertile [HR = 0.70 (95% CI: 0.49-0.99); P-trend = 0.045], whereas no significant associations were found between TDP and overall mortality. TUP is an independent risk factor for mortality among community-dwelling older adults, suggesting that high dietary intake of polyphenols may be associated with longevity.

Scope: Metabolomics approach is focused on identifying all metabolites present in a biological sample (metabolome). Consumption of cocoa products has been related to health benefits including positive effect on cardiovascular health., Methods and Results: Twenty volunteers were included in this randomized, crossover, and controlled clinical trial. After a 2-wk washout period, subjects received 40 g/day of cocoa powder with 500 mL skimmed milk (cocoa with skimmed milk intervention) or 500 mL/day of skimmed milk (skimmed milk intervention) for 4-wk. Urine (24 h) samples were collected at baseline and after each intervention and were analyzed by HPLC-hybrid quadrupole TOF in negative and positive ionization modes followed by multivariate analysis. This analysis revealed a marked separation between the cocoa with skimmed milk intervention and skimmed milk intervention and baseline periods. Thirty-nine compounds linked with cocoa intake, including alkaloid metabolites, polyphenol host and gut microbial metabolites (hydroxyphenylvalerolactones and hydroxyphenylvaleric acids), diketopiperazines and N-phenylpropenoyl-l-amino acids were identified. In the case of endogenous metabolites, putative identifications suggested that metabolites linked with carnitine metabolism and sulfation of tyrosine were decreased by the consumption of cocoa., Conclusion: LC-MS metabolomics strategy allows the defining of a complex metabolic profile derived from cocoa phytochemicals. Likewise, the identification of endogenous markers could lead to new hypotheses to unravel the relationship between cocoa intake and cardiovascular diseases., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: Chronic red wine (RW) consumption has been associated with decreased cardiovascular disease risk, mainly attributed to an improvement in lipid profile. RW intake is also able to change the composition of gut microbiota. High fat intake has recently been reported to increase metabolic endotoxemia. The gut microbiota has been proposed as the main resource of plasma lipopolysaccharides (LPSs) in metabolic endotoxemia., Objective: We analyzed the effect on LPS concentrations of chronic RW consumption and acute RW intake in relation to high fat intake in middle-aged men., Design: For the chronic study, 10 middle-aged male volunteers were randomly assigned in a crossover trial, and after a washout period, all subjects received RW, dealcoholized red wine (DRW), or gin for 20 d. Serum endotoxin and LPS-binding protein (LBP) concentrations were determined after the washout period and after each of the treatments, and changes in fecal microbiota were quantified. For the acute study, 5 adult men underwent a fat overload or a fat overload together with the consumption of RW, DRW, or gin. Baseline and postprandial serum LPS and LBP concentrations and postprandial chylomicron LPS concentrations were measured., Results: There were no significant differences in the change in LPS or LBP concentrations between chronic RW, DRW, and gin consumption. Bifidobacterium and Prevotella amounts were significantly increased by RW and correlated negatively with LPS concentrations. There were no differences in postprandial serum LPS, LBP, or chylomicron LPS concentrations between acute RW, DRW, or gin intake together with a fatty meal., Conclusion: Chronic RW consumption increases Bifidobacterium and Prevotella amounts, which may have beneficial effects by leading to lower LPS concentrations. This trial was registered at controlled-trials.com as ISRCTN88720134.

Background & Aims: Epidemiological data suggest that moderate red wine consumption reduces cardiovascular mortality and the incidence of diabetes. However, whether these effects are due to ethanol or to non-alcoholic components of red wine still remains unknown. The aim of the present study was to compare the effects of moderate consumption of red wine, dealcoholized red wine, and gin on glucose metabolism and the lipid profile., Methods: Sixty-seven men at high cardiovascular risk were randomized in a crossover trial. After a run-in period, all received each of red wine (30 g alcohol/d), the equivalent amount of dealcoholized red wine, and gin (30 g alcohol/d) for 4 week periods, in a randomized order. Fasting plasma glucose and insulin, homeostasis model assessment of insulin resistance (HOMA-IR), plasma lipoproteins, apolipoproteins and adipokines were determined at baseline and after each intervention., Results: Fasting glucose remained constant throughout the study, while mean adjusted plasma insulin and HOMA-IR decreased after red wine and dealcoholized red wine. HDL cholesterol, Apolipoprotein A-I and A-II increased after red wine and gin. Lipoprotein(a) decreased after the red wine intervention., Conclusions: These results support a beneficial effect of the non-alcoholic fraction of red wine (mainly polyphenols) on insulin resistance, conferring greater protective effects on cardiovascular disease to red wine than other alcoholic beverages. www.isrctn.org: ISRCTN88720134., (Copyright © 2012. Published by Elsevier Ltd.)

The natural polyphenol resveratrol has cardiometabolic protective properties. Resveratrol has been reported to be an activator of NAD+-dependent deacetylase sirtuin 1 (SIRT1), which may regulate liver X receptor (LXR) activity, thereby upregulating the expression of genes crucial in reverse cholesterol transport (RCT). In the present study, the effects of resveratrol and SIRT1 overexpression on RCT from macrophages-to-feces in vivo in C57BL/6 mice were determined. [³H]cholesterol-labeled mouse macrophages were injected intraperitoneally into mice treated with intragastric doses of the well-known LXR agonist T0901317, resveratrol, or a vehicle solution, and radioactivity was determined in plasma, liver, and feces. T0901317-treated mice presented increased [³H]cholesterol in plasma and HDL 48 h after the label injection. Treatment with T0901317 also increased liver ABCA1, G1, and G5 gene expression and reduced intestinal cholesterol absorption which were changes that were associated with a 2.8-fold increase in macrophage-derived [³H]cholesterol in feces. In contrast, resveratrol treatment had no effect on liver LXR signaling or fecal [³H]cholesterol excretion. A separate experiment was conducted in SIRT1 transgenic mice. Liver LXR-target gene expression and magnitude of macrophage-derived [³H]cholesterol in plasma, liver, and feces of SIRT1 transgenic mice did not differ from those of wild-type mice. We conclude that neither resveratrol administration nor SIRT1 overexpression upregulate liver LXR-target genes and macrophage-to-feces RCT in vivo., (Copyright © 2013 Mosby, Inc. All rights reserved.)

Blood sample preparation before LC-MS metabolomic fingerprinting is one of the most challenging and error-prone parts of the analytical procedure. Besides proteins, phospholipids contained in blood fluids are known to cause matrix effects and ion suppression phenomena, thus masking biological variation. Nevertheless, the commonly used sample preparation techniques do not consider their removal prior to analysis. Pooled plasma and serum samples were used as biological material, partly as raw samples and partly spiked with distinct concentrations of a metabolite mix (1-5 μg/mL). Prior to LC-ESI-qToF-MS-driven metabolomic analysis, samples were subjected to different preparation methods consisting of three extractions with organic solvents (acetonitrile, methanol, and methanol/ethanol), a membrane-based solvent-free technique, and a hybrid method combining solvent extraction and SPE-mediated removal of phospholipids. The comparative analysis among sample preparation procedures was based on the capacity to detect endogenous compounds in raw samples, differentiate raw versus spiked samples, and reveal real-life metabolomic changes, following a dietary intervention. Method speed, minimum sample handling, compatibility to automation, and applicability to large-scale metabolomic studies were also considered. The combination of solvent deproteinization and the selective removal of phospholipids was revealed to be the most suitable method, in terms of improvement of nonlipid metabolite coverage, extraction reproducibility, quickness, and compatibility with automation, the minimization of matrix effects being among the most probable causes for the good extraction performance associated with the removal of phospholipid species. The main advantage of conventional solvent extraction procedures was the metabolite information coverage for lipid low-molecular-weight species, and extraction with acetonitrile was generally the second choice for sample preparation. Ultrafiltration was the least effective method for plasma and serum preparation; thus, its use without a previous solvent extraction step of the samples should be discarded. According to the presented data, there is no apparent reason to believe that sacrificing information on lipid compounds is too high of a price to pay in order to gain more information on nonlipid LMW metabolites.

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., (Copyright © 2012 Elsevier B.V. All rights reserved.)

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 (t(max)) 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., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Rationale: Experimental studies have shown a potential blood pressure (BP) lowering effect of red wine polyphenols, whereas the effects of ethanol and polyphenols on BP in humans are not yet clear., Objective: The aim of the present work was to evaluate the effects of red wine fractions (alcoholic and nonalcoholic) on BP and plasma nitric oxide (NO) in subjects at high cardiovascular risk., Methods and Results: Sixty-seven men at high cardiovascular risk were studied. After a 2-week run-in period, subjects were randomized into 3 treatment periods in a crossover clinical trial, with a common background diet plus red wine (30g alcohol/day), the equivalent amount of dealcoholized red wine, or gin (30g alcohol/day), lasting 4 weeks each intervention. At baseline and after each intervention, anthropometrical parameters, BP and plasma NO were measured. Systolic and diastolic BP decreased significantly after the dealcoholized red wine intervention and these changes correlated with increases in plasma NO., Conclusions: Dealcoholized red wine decreases systolic and diastolic BP. Our results point out through an NO-mediated mechanism. The daily consumption of dealcoholized red wine could be useful for the prevention of low to moderate hypertension. Trial registered at controlled-trials.com: ISRCTN88720134.

Correctly assessing the metabolic status of subjects after consumption of specific diets is an important challenge for modern nutrition. Recently, metabolomics has been proposed as a powerful tool for exploring the complex relationship between nutrition and health. Nutritional metabolomics, through investigating the role that dietary components play in the maintenance of health and development of risk disease, aims to identify new biomarkers that allow the intake of these compounds to be monitored and related to their expected biological effects. This review offers an overview of the application of nutrimetabolomic strategies in the discovery of new biomarkers in human nutritional research, suggesting three main categories: (1) assessment of nutritional and dietary interventions; (2) diet exposure and food consumption monitoring; and (3) health phenotype and metabolic impact of diet. For this purpose, several examples of these applications will be used to provide evidence and to discuss the advantages and drawbacks of these nutrimetabolomic strategies.

Phenolics are a large group of plant compounds that have been associated with protective health effects against several chronic diseases due to their potential antioxidant, anti-inflammatory, and anticarcinogenic properties. Consequently, in nutritional epidemiology it is essential to make an accurate assessment of phenolic exposure to evaluate their protective activities against human diseases. Self-reported questionnaires and biomarkers are the two main methods used for estimating dietary phenolics. Despite the limitations of self-reported methods, they are still an acceptable and useful measure. Meanwhile, nutritional biomarkers provide an alternative, more accurate measure, but they are expensive, and to date there are few validated biomarkers of phenolic intake. Nowadays, new analytical techniques, using both targeted and untargeted metabolomic approaches, play an important part in the rapid increase in the understanding of phenolic bioavailability and, consequently, have provided new potential biomarkers in small trials. In the near future, these dietary biomarkers should be tested in large epidemiological studies. Furthermore, the use of two independent measures-questionnaires and biomarkers-together provides a more thorough analysis of true phenolic exposure. Indeed, the challenge in the long term is to combine the information from biomarkers and self-reported questionnaires to clarify the relationship between dietary phenolics and disease.

Adherence to a Mediterranean diet (MD) is associated with a reduced risk of coronary heart disease. However, the molecular mechanisms involved are not fully understood. The aim of this study was to compare the effects of 2 MD with those of a low-fat-diet (LFD) on circulating inflammatory biomarkers related to atherogenesis. A total of 516 participants included in the Prevention with Mediterranean Diet Study were randomized into 3 intervention groups [MD supplemented with virgin olive oil (MD-VOO); MD supplemented with mixed nuts (MD-Nuts); and LFD]. At baseline and after 1 y, participants completed FFQ and adherence to MD questionnaires, and plasma concentrations of inflammatory markers including intercellular adhesion molecule-1(ICAM-1), IL-6, and 2 TNF receptors (TNFR60 and TNFR80) were measured by ELISA. At 1 y, the MD groups had lower plasma concentrations of IL-6, TNFR60, and TNFR80 (P < 0.05), whereas ICAM-1, TNFR60, and TNFR80 concentrations increased in the LFD group (P < 0.002). Due to between-group differences, participants in the 2 MD groups had lower plasma concentrations of ICAM-1, IL-6, TNFR60, and TNFR80 compared to those in the LFD group (P ≤ 0.028). When participants were categorized in tertiles of 1-y changes in the consumption of selected foods, those in the highest tertile of virgin olive oil (VOO) and vegetable consumption had a lower plasma TNFR60 concentration compared with those in tertile 1 (P < 0.02). Moreover, the only changes in consumption that were associated with 1-y changes in the geometric mean TNFR60 concentrations were those of VOO and vegetables (P = 0.01). This study suggests that a MD reduces TNFR concentrations in patients at high cardiovascular risk.

Moderate wine consumption has been shown to reduce cardiovascular (CV) risk, due to alcohol and polyphenolic compounds, such as resveratrol. We investigated the associations between total urinary resveratrol metabolites (TRMs) as biomarkers of wine and resveratrol consumption and CV risk factors in a large cross-sectional study including high CV risk individuals in Spain. We studied 1000 participants in the PREDIMED Study in whom TRMs were analyzed by LC-MS/MS with a previous solid phase extraction. Multiple linear regression of TRMs (biomarker of wine consumption) improved the mean (95% CI) of HDL [0.168 (0.027-0.309); P=0.02] and triglyceride [-1.012 (-1.797 to -0.227); P=0.012] plasma concentrations and heart rate [-0.259 (-0.412 to -0.107); P<0.001]. Models of TRMs adjusted for alcohol (biomarker of resveratrol intake) decreased fasting blood glucose [-0.533 (-1.034 to -0.033); P=0.037] and triglyceride [-1.014 (-1.998 to -0.029); P=0.044] concentrations, and heart rate [-0.277 (-0.467 to -0.087); P=0.004]. Both resveratrol and wine intake, evaluated as TRMs, were associated with beneficial changes in blood lipid profiles, fasting blood glucose (only resveratrol) and heart rate, suggesting that resveratrol intake via wine consumption might help to decrease CV risk factors., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Previous epidemiological and feeding studies have observed that adherence to Mediterranean diet (Med-Diet) is associated with reduced cardiovascular risk. However, the molecular mechanisms involved are not fully understood. Since atherosclerosis is nowadays considered a low-grade inflammatory disease, recent studies have explored the anti-inflammatory effects of a Med-Diet intervention on serum and cellular biomarkers related to atherosclerosis. In two sub-studies of the PREDIMED (PREvencion con DIeta MEDiterranea) trial, we analyzed the effects at 3 months of two Med-Diet interventions supplemented with either virgin olive oil (VOO) or nuts compared with a control low-fat diet (LFD). Both Med-Diets showed an anti-inflammatory effect reducing serum C-reactive protein, interleukin-6 (IL6) and endothelial and monocytary adhesion molecules and chemokines (P<0.05; all), whereas these parameters increased after the LFD intervention (P<0.05; all). In another substudy, we evaluated the long-term (1 year) effects of these interventions on vascular risk factors in 516 high-risk subjects, as well as the effect of different Med-Diet components in the reduction of these biomarkers. At 1 year, the Med-Diet groups had significant decreases in the plasma concentrations of IL6, tumor necrosis factor receptor (TNFR) 60 and TNFR80 (P<0.05), while intercellular adhesion molecule 1 (ICAM-1), TNFR60 and TNFR80 concentrations increased in the LFD group (P<0.002). In addition, those allocated in the highest tertile of VOO and vegetables consumption had a significant diminution of plasma TNFR60 concentration compared with those in tertile 1 (P<0.02). In conclusion, Med-Diet exerts an anti-inflammatory effect on cardiovascular system since it down-regulates cellular and circulating inflammatory biomarkers related to atherogenesis in subjects at high cardiovascular risk., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

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.

Background: Few clinical studies have focused on the alcohol-independent cardiovascular effects of the phenolic compounds of red wine (RW)., Objective: We 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., Design: Sixty-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., Results: Alcohol 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., Conclusion: The 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.

Polyphenols have beneficial effects on several chronic diseases but assessing polyphenols intake from self-reported dietary questionnaires tends to be inaccurate and not very reliable. A promising alternative is to use urinary excretion of polyphenols as a proxy measure of intake. The best method to assess urinary excretion is to collect 24-h urine. However, since collecting 24-h urine method is expensive, time consuming and may be difficult to implement in large population-based studies, measures obtained from spot urine normalized by creatinine are commonly used. The purpose of the study was to evaluate the correlation between polyphenols dietary intake and total urinary polyphenol excretion (TPE), expressed by both 24-h volume and urinary creatinine normalization in 928 participants from the InCHIANTI study. Dietary intake data were collected using a validated food frequency questionnaire. Urinary TPE was analyzed by Folin-Ciocalteau assay. Both urinary TPE expression models were statistically correlated (r=0.580), and the partial correlation coefficient improved (pr=0.722) after adjusting for the variables that modify the urinary creatinine excretion (i.e. gender, age, BMI, physical activity and renal function). In crude models, polyphenol intake was associated with TPE corrected by 24-h volume (r=0.211; P<0.001), but not with creatinine normalization (r=0.014; P=0.692). However, urinary TPE expressed by creatinine correction was significantly correlated with dietary polyphenols after adjusting for covariates (pr=0.113; P=0.002). We conclude that urinary TPE expressed by 24-h volume is a better biomarker of polyphenol dietary intake than by urinary creatinine normalization. After covariate adjustment, both can be used for studying the relationships between polyphenol intake and health in large-scale epidemiological studies., Competing Interests: The authors are not aware of any conflict of interest., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The lay press often heralds polyphenols as panacea for all sorts of diseases. The rationale is that their antioxidant activity would prevent free radical damage to macromolecules. However, basic and clinical science is showing that the reality is much more complex than this and that several issues, notably content in foodstuff, bioavailability, or in vivo antioxidant activity are yet to be resolved. We summarize the recent findings concerning the effects of polyphenols on human health, analyze the current limitations at pitfalls, and propose future directions for research.

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.

Tomatoes and tomato-based products are rich in antioxidants such as carotenoids, vitamin C and polyphenols. The industrial processing of diced tomatoes involves heat treatments in which these antioxidant compounds may be potentially affected. In this study, we evaluate the effect of each separate step in the dice-making process. Three technological processes were investigated: Hot, Cold and Cold treated with calcium salt (CaCl2). Four stages were monitored in each process: (1) fresh tomatoes; (2) peeled tomatoes; (3) diced tomatoes; and (4) final product after sauce addition. The main tool for minimising or counteracting the eventual processing damage was the strategy of 'reconstitution', achieved by adding a sauce rich in seeds and peels with high levels of antioxidants and phenolics to the diced tomatoes. Different analyses were carried out in order to evaluate the effect of each processing step. First, total polyphenols (TP) were evaluated using Folin-Ciocalteau (F-C) assay and antioxidant activity using ABTS(+) and DPPH assays. Flavonols, flavanones, hydroxycinnamic and phenolic acids were then quantified using liquid chromatography/electrospray ionisation tandem mass spectrometry (HPLC-ESI-MS/MS). The combination of principal component analysis (PCA) and analysis of variance (ANOVA) revealed that each processing step induces alterations in the antioxidant and phenolic profile, and in particular sauce addition and calcium treatment significantly affected the levels of antioxidants and phenolics during the dice-making process., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Considerable information on the chemistry and biological properties of dietary phytochemicals has accumulated over the past three decades. The scattering of the data in tens of thousands publications and the diversity of experimental approaches and reporting formats all make the exploitation of this information very difficult. Some of the data have been collected and stored in electronic databases so that they can be automatically updated and retrieved. These databases will be particularly important in the evaluation of the effects on health of phytochemicals and in facilitating the exploitation of nutrigenomic data. The content of over 50 databases on chemical structures, spectra, metabolic pathways in plants, occurrence and concentrations in foods, metabolism in humans and animals, biological properties, and effects on health or surrogate markers of health is reviewed. Limits of these databases are emphasized, and needs and recommendations for future developments are underscored. More investments in the construction of databases on phytochemicals and their effects on health are clearly needed. They should greatly contribute to the success of future research in this field.

Tomatoes (Solanum lycopersicum L.), the second most important vegetable crop worldwide, are a key component in the so-called "Mediterranean diet", which is strongly associated with a reduced risk of chronic degenerative diseases. In this work, we evaluate the differences in the total and individual polyphenol content and hydrophilic antioxidant capacity of seven varieties of tomato cultivated in Vegas Bajas del Guadiana, Badajoz (Spain), which were collected from two consecutive harvests (2008-2009). Hydrophilic antioxidant capacity was evaluated using the TEAC assay, while the Folin-Ciocalteau assay with a previous cleanup was used to establish total polyphenol content. The method was optimized and validated. Individual polyphenols were quantified using liquid chromatography/electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) on a triple quadrupole. All compounds were found to be significantly different when analysis of variance was performed. Results from the principal component analysis show that phenolic compounds and hydrophilic antioxidant capacity were responsible for the differences among tomato samples according to variety.

Polyphenols exert beneficial effects in atherosclerosis. The crucial step in atherosclerosis is the recruitment of monocytes to the subendothelial space, induced by endothelial adhesion molecules through the activation of factors such as NF-κB. We studied the effect of a dealcoholised lager beer (DLB) and a dealcoholised dark beer (DDB) on atherosclerotic lesions, and the underlying mechanisms. Dealcoholised beers were administered in the diet (42 ml/kg body weight per d) to 4-week-old male apoE knockout (apoE - / - ) mice for 20 weeks. The atherosclerotic lesions in the thoracic aorta were reduced by 44 % (P = 0·003) and 51 % (P < 0·001) in DLB- and DDB-treated mice, respectively. Also, the mRNA expressions of the endothelial adhesion molecules in the total aorta were decreased: P-selectin showed a 17 % (P = 0·004) reduction in DDB-treated mice; vascular cell adhesion molecule-1 (VCAM-1) was decreased by 20 % (P = 0·012) and 32 % (P = 0·001) in DLB- and DDB-treated mice, respectively; intercellular adhesion molecule-1 (ICAM-1) showed a 14 % (P = 0·014) reduction in DLB-treated mice. The protein expressions of these molecules and NF-κB were studied in the aortic root. P-selectin was decreased by 37 % (P = 0·012) in DDB-treated mice; VCAM-1 was reduced by 48 % (P = 0·001) and 54 % (P < 0·001) in DLB- and DDB-treated mice, respectively; ICAM-1 was decreased by 25 % (P = 0·028) and 30 % (P = 0·018) in DLB- and DDB-treated mice, respectively; NF-κB was reduced by 46 % (P = 0·042) in DDB-treated mice. In conclusion, dealcoholised beers protected apoE - / -  mice against atherosclerosis, through the modulation of endothelial adhesion molecules, possibly induced by NF-κB.

Flavan-3-ols, occurring in monomeric, as well as in oligomeric and polymeric forms (also known as condensed tannins or proanthocyanidins), are among the most abundant and bioactive dietary polyphenols, but their in vivo health effects in humans may be limited because of their recognition as xenobiotics. Bioavailability of flavan-3-ols is largely influenced by their degree of polymerization; while monomers are readily absorbed in the small intestine, oligomers and polymers need to be biotransformed by the colonic microbiota before absorption. Therefore, phenolic metabolites, rather than the original high molecular weight compounds found in foods, may be responsible for the health effects derived from flavan-3-ol consumption. Flavan-3-ol phenolic metabolites differ in structure, amount and excretion site. Phase II or tissular metabolites derived from the small intestine and hepatic metabolism are presented as conjugated derivatives (glucuronic acid or sulfate esters, methyl ether, or their combined forms) of monomeric flavan-3-ols and are preferentially eliminated in the bile, whereas microbial metabolites are rather simple conjugated lactones and phenolic acids that are largely excreted in urine. Although the colon is seen as an important organ for the metabolism of flavan-3-ols, the microbial catabolic pathways of these compounds are still under consideration, partly due to the lack of identification of bacteria with such capacity. Studies performed with synthesized or isolated phase II conjugated metabolites have revealed that they could have an effect beyond their antioxidant properties, by interacting with signalling pathways implicated in important processes involved in the development of diseases, among other bioactivities. However, the biological properties of microbe-derived metabolites in their actual conjugated forms remain largely unknown. Currently, there is an increasing interest in their effects on intestinal infections, inflammatory intestinal diseases and overall gut health. The present review will give an insight into the metabolism and microbial biotransformation of flavan-3-ols, including tentative catabolic pathways and aspects related to the identification of bacteria with the ability to catabolize these kinds of polyphenols. Also, the in vitro bioactivities of phase II and microbial phenolic metabolites will be covered in detail.

Almond, as a part of the nut family, is an important source of biological compounds, and specifically, almond skins have been considered an important source of polyphenols, including flavan-3-ols and flavonols. Polyphenol metabolism may produce several classes of metabolites that could often be more biologically active than their dietary precursor and could also become a robust new biomarker of almond polyphenol intake. In order to study urinary metabolome modifications during the 24 h after a single dose of almond skin extract, 24 volunteers (n = 24), who followed a polyphenol-free diet for 48 h before and during the study, ingested a dietary supplement of almond skin phenolic compounds (n = 12) or a placebo (n = 12). Urine samples were collected before ((-2)-0 h) and after (0-2 h, 2-6 h, 6-10 h, and 10-24 h) the intake and were analyzed by liquid chromatography-mass spectrometry (LC-q-TOF) and multivariate statistical analysis (principal component analysis (PCA) and orthogonal projection to latent structures (OPLS)). Putative identification of relevant biomarkers revealed a total of 34 metabolites associated with the single dose of almond extract, including host and, in particular, microbiota metabolites. As far as we know, this is the first time that conjugates of hydroxyphenylvaleric, hydroxyphenylpropionic, and hydroxyphenylacetic acids have been identified in human samples after the consumption of flavan-3-ols through a metabolomic approach. The results showed that this non-targeted approach could provide new intake biomarkers, contributing to the development of the food metabolome as an important part of the human urinary metabolome.

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.

Nut skins are considered to be a rich source of polyphenols and may be partially responsible for the numerous health effects associated with nut consumption. However, more bioavailability studies of nut skin polyphenols are needed to understand the health effects derived from nut consumption. The aim of the present study was to determine the profiles of both phase II and microbial-derived phenolic metabolites in plasma and urine samples before and after the intake of almond skin polyphenols by healthy human subjects (n = 2). Glucuronide, O-methyl glucuronide, sulfate, and O-methyl sulfate derivatives of (epi)catechin, as well as the glucuronide conjugates of naringenin and glucuronide and sulfate conjugates of isorhamnetin, were detected in plasma and urine samples after consumption of almond skin polyphenols. The main microbial-derived metabolites of flavanols, such as 5-(dihydroxyphenyl)-gamma-valerolactone and 5-(hydroxymethoxyphenyl)-gamma-valerolactone, were also detected in their glucuronide and sulfate forms. In addition, numerous metabolites derived from further microbial degradation of hydroxyphenylvalerolactones, including hydroxyphenylpropionic, hydroxyphenylacetic, hydroxycinnamic, hydroxybenzoic, and hydroxyhippuric acids, registered major changes in urine after the consumption of almond skin polyphenols. The urinary excretion of these microbial metabolites was estimated to account for a larger proportion of the total polyphenol ingested than phase II metabolites of (epi)catechin, indicating the important role of intestinal bacteria in the metabolism of highly polymerized almond skin polyphenols. To the authors' knowledge this study constitutes the most complete report of the absorption of almond skin polyphenols in humans.

Cocoa-phytochemicals have been related to the health-benefits of cocoa consumption. Metabolomics has been proposed as a powerful tool to characterize both the intake and the effects on the metabolism of dietary components. Human urine metabolome modifications after single cocoa intake were explored in a randomized, crossed, and controlled trial. After overnight fasting, 10 subjects consumed randomly either a single dose of cocoa powder with milk or water, or milk without cocoa. Urine samples were collected before the ingestion and at 0-6, 6-12, and 12-24-h after test-meals consumption. Samples were analyzed by HPLC-q-ToF, followed by multivariate data analysis. Results revealed an important effect on urinary metabolome during the 24 h after cocoa powder intake. These changes were not influenced by matrix as no global differences were found between cocoa powder consumption with milk or with water. Overall, 27 metabolites related to cocoa-phytochemicals, including alkaloid derivatives, polyphenol metabolites (both host and microbial metabolites) and processing-derived products such as diketopiperazines, were identified as the main contributors to the urinary modifications after cocoa powder intake. These results confirm that metabolomics will contribute to better characterization of the urinary metabolome in order to further explore the metabolism of phytochemicals and its relation with human health.

Background: Epidemiologic studies have suggested that flavonoid intake plays a critical role in the prevention of coronary heart disease. Because atherosclerosis is considered a low-grade inflammatory disease, some feeding trials have analyzed the effects of cocoa (an important source of flavonoids) on inflammatory biomarkers, but the results have been controversial., Objective: The objective was to evaluate the effects of chronic cocoa consumption on cellular and serum biomarkers related to atherosclerosis in high-risk patients., Design: Forty-two high-risk volunteers (19 men and 23 women; mean +/- SD age: 69.7 +/- 11.5 y) were included in a randomized crossover feeding trial. All subjects received 40 g cocoa powder with 500 mL skim milk/d (C+M) or only 500 mL skim milk/d (M) for 4 wk. Before and after each intervention period, cellular and serum inflammatory biomarkers related to atherosclerosis were evaluated., Results: Adherence to the dietary protocol was excellent. No significant changes in the expression of adhesion molecules on T lymphocyte surfaces were found between the C+M and M groups. However, in monocytes, the expression of VLA-4, CD40, and CD36 was significantly lower (P = 0.005, 0.028, and 0.001, respectively) after C+M intake than after M intake. In addition, serum concentrations of the soluble endothelium-derived adhesion molecules P-selectin and intercellular adhesion molecule-1 were significantly lower (both P = 0.007) after C+M intake than after M intake., Conclusions: These results suggest that the intake of cocoa polyphenols may modulate inflammatory mediators in patients at high risk of cardiovascular disease. These antiinflammatory effects may contribute to the overall benefits of cocoa consumption against atherosclerosis. This trial was registered in the Current Controlled Trials at London, International Standard Randomized Controlled Trial Number, at controlled-trials.com as ISRCTN75176807.

Proanthocyanidins, flavonoids exhibiting cardiovascular protection, constitute a major fraction of the flavonoid ingested in the human diet. Although they are poorly absorbed, they are metabolized by the intestinal microbiota into various phenolic acids. An analytical method, based on an optimized 96-well plate solid-phase extraction (SPE) procedure and liquid chromatography tandem mass spectrometry (SPE-LC-MS/MS) for the analysis of 19 phenolic microbial metabolites and monomeric and dimeric flavanols in urine samples, was developed and validated. Human urine samples were obtained before and after ingestion of an acute consumption of 40 g of soluble cocoa powder and rat urines before and after the prolonged administration (2 weeks) of different diets composed of natural cocoa powder. The mean recovery of analytes using the new SPE-LC-MS/MS method ranged from 87% to 109%. Accuracy ranged from 87.5% to 113.8%, and precision met acceptance criteria (<15% relative standard deviation). Procyanidin B2 has been detected and quantified for the first time in human and rat urine after cocoa consumption. Changes in human and rat urinary levels of microbial phenolic acids and flavanols were in the range of 0.001-59.43 nmol/mg creatinine and of 0.004-181.56 nmol/mg creatinine, respectively. Major advantages of the method developed include reduction of laboratory work in the sample preparation step by the use of 96-well SPE plates and the sensitive measurement of a large number of metabolites in a very short run time, which makes it ideal for use in epidemiological studies.

Several clinical and epidemiological studies have shown that moderate wine consumption may exert a protective effect against oxidative stress involved in several diseases, such as cardiovascular and neurodegenerative disorders. However, the epidemiological assessment of wine consumption has usually been obtained using self-reported questionnaires containing less reliable information for assessing total intake than nutritional biomarkers. A reliable biomarker for wine consumption is, therefore, needed. To validate urinary resveratrol metabolites (RMs) as a biomarker of wine consumption in a large cohort of free-living subjects, 1000 consecutive subjects entering a substudy of the PREDIMED trial (Prevención con Dieta Mediterránea) were evaluated. Data were collected in a validated semiquantitative food frequency questionnaire. RMs were measured in morning urine by LC-MS/MS. Urinary RM values correlated directly with reported daily amounts of wine consumed (r=0.895; p<0.001). One drink of wine per week can be detected. Using a cut-off of 411.4 nmol/g creatinine, the measurement of urinary RMs could discriminate wine consumers from non-wine consumers with a sensitivity of 93.3% (95% confidence interval (CI) 91.5-94.7%) and a specificity of 92.1% (CI 90.2-93.7%). Urinary RMs fulfill the criteria to be considered as a nutritional biomarker of wine consumption in a large sample of free-living subjects. This biomarker would provide an additional tool for investigating more precisely the relationship between wine consumption and health benefits.

Background: The urinary flavonoids are considered a reliable biomarker for the intake of polyphenol-rich foods., Objectives: To assess the normal distribution of urinary polyphenol [PP] excretion among healthy male children and adolescents on a typical Egyptian diet. To follow up the impact of nutritional intervention with tomato juice on the urinary excretion of [PP]., Subjects: Forty-nine male subjects 7-14 years old collected a 24-h urine sample and filled a dietary record during a 7-day period. A daily serving of 230 g fresh tomato juice was followed for 18 days in a subgroup. Total urinary [PP] excretions were measured before and after termination of the intervention program. The total urinary [PP] was analyzed after a clean-up solid-phase extraction step by the Folin-Ciocalteu reagent in the 96 micro plates. The results were expressed as gallic acid equivalents (GAE)., Results: The urinary [PP] excretion averaged 48.6+/-5.5 mg GAE/24 h, equivalent to 89.5+/-8.4 mg GAE/g creatinine. The mean urinary [PP] excretion increased significantly (P<0.05) following the intervention with tomato juice (287.4+/-64.3 mg GAE/g creatinine) compared with the respective mean baseline level (94.5+/-8.92 mg GAE/g creatinine)., Conclusion: Clinical laboratory reference limits for urinary polyphenols are presented for Egyptian male children and adolescents. Measuring the urinary polyphenol excretion proved a good biomarker for the dietary polyphenol intake and the results demonstrated that tomato [PP] was highly bioavailable in the human body.

Nutritional markers have several advantages for epidemiologic and clinical assays, when compared to dietary data obtained by food frequency questionnaires. Few studies have assessed whether total polyphenol (TP) compounds provide a valid biomarker for TP intake. To date, there has been almost no literature describing methods to determine TP in complex matrices such as urine, which have many interfering substances. We report a rapid Folin-Ciocalteu method to determine TP in urine samples using Oasis((R)) MAX 96-well plate cartridges for solid phase extraction. These plates allow analysis of a high number of samples at the same time. We performed a prospective, randomized, crossover trial and one cross-sectional study with 60 volunteers from the PREDIMED trial, seeking to evaluate whether the TP in urine were correlated with polyphenol intake and could, therefore, be considered as a marker of intake of these compounds. The assay was optimized; the sensitivity and the polarity range of urine polyphenols were increased and the detection and quantification limits were significantly reduced. The metabolites in standards solution and urine samples were stable under the storage and handling conditions. In the clinical trial and the cross-sectional study, TP excreted in spot urine samples were positively correlated with TP intake, r=0.48, P<0.01 and r=0.257, P=0.04, respectively. The methodology described may be used to detect TP in urine samples, employing the high throughput of 96-well microtiter plates and reader. The method is fast and simple and it allows analysis of a large number of samples at the same time.

Due to supporting evidence that dietary patterns may have a significant role in the maintenance of good physical performance with aging, we tested whether plasma fatty acids, saturated fatty acids (SFA), and polyunsaturated (PUFA) fatty acids are cross-sectionally associated with different physical performance and predict changes in physical performance over a 3-year period. Data were from the InCHIANTI study, a population-based study of older Italians. Plasma fatty acids were measured at enrollment (1998-2000), and outcome variables, Summary Physical Performance Battery (SPPB), and time to walk 7 meters (m) were measured at enrollment and after 3 years (2001-2004). At enrollment, 330 participants had significantly impaired lower extremity performance (defined as a SPPB score < or = 9). Adjusting for age, participants with a SPPB score > 9 had higher levels of total PUFA, n-3 PUFA, and n-6 PUFA, while significantly lower levels of SFA than those with a SPPB score < 9. Baseline SPPB scores were also associated with n-3 PUFA (beta = 0.148, p = 0.031), whereas the 7-m walk time was associated with total PUFA (beta = - 0.068, p = 0.008), after adjusting for potential confounders. Of the 884 participants with a SPPB score > 9 at baseline, 114 (12.9%) developed impaired lower extremity performance (SPPB < or = 9). In fully adjusted logistic models, baseline n-3 PUFA levels were inversely related to the risk of developing a decline in SPPB to < or = 9 (odds ratio [OR] = 0.21; 95% confidence interval [CI] = 0.08-0.53), while the n-6/n-3 ratio was associated with a higher risk of SPPB decline to < or = 9 (OR = 5.23; 95% CI = 2.02-13.51). In multivariate regression models, the n-6/n-3 ratio was associated with a longer time to walk 7 m (beta = 0.396, p = 0.037). n-3 PUFA plasma levels, which most likely reflect dietary intake, seem to protect against accelerated decline of physical performance. A higher n-6/n-3 ratio was associated with higher risk of developing poor physical performance and slower walking speed.