Characterization of the Human Exposome by a Comprehensive and Quantitative Large-Scale Multianalyte Metabolomics Platform.

The exposome, defined as the cumulative measure of external exposures and associated biological responses throughout the lifespan, has emerged in recent years as a cornerstone in biomedical sciences. Metabolomics stands out here as one of the most powerful tools for investigating the interplay between the genetic background, exogenous, and endogenous factors within human health. However, to address the complexity of the exposome, novel methods are needed to characterize the human metabolome. In this work, we have optimized and validated a multianalyte metabolomics platform for large-scale quantitative exposome research in plasma and urine samples, based on the use of simple extraction methods and high-throughput metabolomic fingerprinting. The methodology enables, for the first time, the simultaneous characterization of the endogenous metabolome, food-related metabolites, pharmaceuticals, household chemicals, environmental pollutants, and microbiota derivatives, comprising more than 1000 metabolites in total. This comprehensive and quantitative investigation of the exposome is achieved in short run times, through simple extraction methods requiring small-sample volumes, and using integrated quality control procedures for ensuring data quality. This metabolomics approach was satisfactorily validated in terms of linearity, recovery, matrix effects, specificity, limits of quantification, intraday and interday precision, and carryover. Furthermore, the clinical potential of the methodology was demonstrated in a dietary intervention trial as a case study. In summary, this study describes the optimization, validation, and application of a multimetabolite platform for comprehensive and quantitative metabolomics-based exposome research with great utility in large-scale epidemiological studies.

Quantifying the human diet in the crosstalk between nutrition and health by multi-targeted metabolomics of food and microbiota-derived metabolites.

BACKGROUND: Metabolomics is a powerful tool for investigating the association between nutrition and health status. Although urine is commonly employed for studying the metabolism and transformation of food components, the use of blood samples could be preferable to gain new insights into the bioavailability of diet-derived compounds and their involvement in health. However, the chemical complexity of blood samples hinders the analysis of this biological fluid considerably, which makes the development of novel and comprehensive analytical methods mandatory. METHODS: In this work, we optimized a multi-targeted metabolomics platform for the quantitative and simultaneous analysis of 450 food-derived metabolites by ultra-high performance liquid chromatography coupled to tandem mass spectrometry. To handle the chemical complexity of blood samples, three complementary extraction methods were assayed and compared in terms of recovery, sensitivity, precision and matrix effects with the aim of maximizing metabolomics coverage: protein precipitation, reversed solid-phase extraction, and hybrid protein precipitation with solid-phase extraction-mediated phospholipid removal. RESULTS: After careful optimization of the extraction conditions, protein precipitation enabled the most efficient and high-throughput extraction of the food metabolome in plasma, although solid-phase extraction-based protocols provided complementary performance for the analysis of specific polyphenol classes. The developed method yielded accurate recovery rates with negligible matrix effects, and good linearity, as well as high sensitivity and precision for most of the analyzed metabolites. CONCLUSIONS: The multi-targeted metabolomics platform optimized in this work enables the simultaneous detection and quantitation of 450 dietary metabolites in short-run times using small volumes of biological sample, which facilitates its application to epidemiological studies.

Chronic adenosine A2A receptor blockade induces locomotor sensitization and potentiates striatal LTD IN GPR37-deficient mice.

Adenosine A2A receptors (A2A R) play a key role in modulating dopamine-dependent locomotor activity, as heralded by the sensitization of locomotor activity upon chronic A2A R blockade, which is associated with elevated dopamine levels and altered corticostriatal synaptic plasticity. Since the orphan receptor GPR37 has been shown to modulate A2A R function in vivo, we aimed to test whether the A2A R-mediated sensitization of locomotor activity is GPR37-dependent and involves adaptations of synaptic plasticity. To this end, we administered a selective A2A R antagonist, SCH58261 (1 mg/kg, i.p.), daily for 14 days, and the locomotor sensitization, striatum-dependent cued learning, and corticostriatal synaptic plasticity (i.e., long-term depression) were compared in wild-type and GPR37-/- mice. Notably, GPR37 deletion promoted A2A R-associated locomotor sensitization but not striatum-dependent cued learning revealed upon chronic SCH58261 treatment of mice. Furthermore, chronic A2A R blockade potentiated striatal long-term depression in corticostriatal synapses of GPR37-/- but not of wild-type mice, thus correlating well with neurochemical alterations of the adenosinergic system. Overall, these results revealed the importance of GPR37 regulating A2A R-dependent locomotor sensitization and synaptic plasticity in the basal ganglia circuitry. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/. Open Science: This manuscript was awarded with the Open Materials Badge. For more information see: https://cos.io/our-services/open-science-badges/.

Characterization of Metabolomic Profile Associated with Metabolic Improvement after Bariatric Surgery in Subjects with Morbid Obesity.

The exact impact of bariatric surgery in metabolically "healthy" (MH) or "unhealthy" (MU) phenotypes for the study of the metabolic improvement is still unknown. We applied an untargeted LC-ESI-TripleTOF-MS-driven metabolomics approach in serum samples from 39 patients with morbid obesity (MH and MU) 1, 3, and 6 months after bariatric surgery. Multiple factor analysis, along with correlation and enrichment analyses, was carried out to distinguish those metabolites associated with metabolic improvement. Hydroxypropionic acids, medium-/long-chain hydroxy fatty acids, and bile acid glucuronides were the most discriminative biomarkers of response between MH and MU phenotypes. Hydroxypropionic (hydroxyphenyllactic-related) acids, amino acids, and glycerolipids were the most significant clusters of metabolites altered after bariatric surgery in MU ( p < 0.001). After surgery, MU and MH changed toward a common metabolic state 3 months after surgery. We observed a negative correlation with changes in waist circumference and cholesterol levels with metabolites of lipid metabolism. Glycemic variables were correlated with hexoses, which, in turn, correlated with gluconic acid and amino acid metabolism. Finally, we noted that hydroxyphenyllactic acid was associated with amino acid and lipid metabolism. Microbial metabolism of amino acid and BA glucuronidation pathways may be the key points of metabolic rearrangement after surgery.

Untargeted Profiling of Concordant/Discordant Phenotypes of High Insulin Resistance and Obesity To Predict the Risk of Developing Diabetes.

This study explores the metabolic profiles of concordant/discordant phenotypes of high insulin resistance (IR) and obesity. Through untargeted metabolomics (LC-ESI-QTOF-MS), we analyzed the fasting serum of subjects with high IR and/or obesity ( n = 64). An partial least-squares discriminant analysis with orthogonal signal correction followed by univariate statistics and enrichment analysis allowed exploration of these metabolic profiles. A multivariate regression method (LASSO) was used for variable selection and a predictive biomarker model to identify subjects with high IR regardless of obesity was built. Adrenic acid and a dyglyceride (DG) were shared by high IR and obesity. Uric and margaric acids, 14 DGs, ketocholesterol, and hydroxycorticosterone were unique to high IR, while arachidonic, hydroxyeicosatetraenoic (HETE), palmitoleic, triHETE, and glycocholic acids, HETE lactone, leukotriene B4, and two glutamyl-peptides to obesity. DGs and adrenic acid differed in concordant/discordant phenotypes, thereby revealing protective mechanisms against high IR also in obesity. A biomarker model formed by DGs, uric and adrenic acids presented a high predictive power to identify subjects with high IR [AUC 80.1% (68.9-91.4)]. These findings could become relevant for diabetes risk detection and unveil new potential targets in therapeutic treatments of IR, diabetes, and obesity. An independent validated cohort is needed to confirm these results.

Mechanistically different effects of fat and sugar on insulin resistance, hypertension, and gut microbiota in rats.

Insulin resistance (IR) and impaired glucose tolerance (IGT) are the first manifestations of diet-induced metabolic alterations leading to Type 2 diabetes, while hypertension is the deadliest risk factor of cardiovascular disease. The roles of dietary fat and fructose in the development of IR, IGT, and hypertension are controversial. We tested the long-term effects of an excess of fat or sucrose (fructose/glucose) on healthy male Wistar-Kyoto (WKY) rats. Fat affects IR and IGT earlier than fructose through low-grade systemic inflammation evidenced by liver inflammatory infiltration, increased levels of plasma IL-6, PGE2, and reduced levels of protective short-chain fatty acids without triggering hypertension. Increased populations of gut Enterobacteriales and Escherichia coli may contribute to systemic inflammation through the generation of lipopolysaccharides. Unlike fat, fructose induces increased levels of diacylglycerols (lipid mediators of IR) in the liver, urine F2-isoprostanes (markers of systemic oxidative stress), and uric acid, and triggers hypertension. Elevated populations of Enterobacteriales and E. coli were only detected in rats given an excess of fructose at the end of the study. Dietary fat and fructose trigger IR and IGT in clearly differentiated ways in WKY rats: early low-grade inflammation and late direct lipid toxicity, respectively; gut microbiota plays a role mainly in fat-induced IR, and hypertension is independent of inflammation-mediated IR. The results provide evidence that suggests that the combination of fat and sugar is potentially more harmful than fat or sugar alone when taken in excess.

New and vintage solutions to enhance the plasma metabolome coverage by LC-ESI-MS untargeted metabolomics: the not-so-simple process of method performance evaluation.

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.

Effect of n-3 PUFA supplementation at different EPA:DHA ratios on the spontaneously hypertensive obese rat model of the metabolic syndrome.

The increasing incidence of the metabolic syndrome (MetS), a combination of risk factors before the onset of CVD and type 2 diabetes, encourages studies on the role of functional food components such as long-chain n-3 PUFA as preventive agents. In the present study, we explore the effect of EPA and DHA supplementation in different proportions on spontaneously hypertensive obese (SHROB) rats, a model for the MetS in a prediabetic state with mild oxidative stress. SHROB rats were randomised into four groups (n 7), each supplemented with EPA/DHA at ratios of 1:1, 2:1 and 1:2, or soyabean oil as the control for 13 weeks. The results showed that in all the proportions tested, EPA/DHA supplementation significantly lowered total and LDL-cholesterol concentrations, compared with those of the control group. EPA/DHA supplementation at the ratios of 1:1 and 2:1 significantly decreased inflammation (C-reactive protein levels) and lowered oxidative stress (decreased excretion of urinary isoprostanes), mainly at the ratio of 1:2. The activity of antioxidant enzymes increased in erythrocytes, abdominal fat and kidneys, with magnitudes depending on the EPA:DHA ratio. PUFA mixtures from fish affected different MetS markers of CVD risk factors in SHROB rats, depending on the ratios of EPA/DHA supplementation. The activation of endogenous defence systems may be related to the reduction of inflammation and oxidative stress.

Sensitive and Rapid UHPLC-MS/MS for the Analysis of Tomato Phenolics in Human Biological Samples.

An UHPLC-MS/MS method for the quantification of tomato phenolic metabolites in human fluids was optimized and validated, and then applied in a pilot dietary intervention study with healthy volunteers. A 5-fold gain in speed (3.5 min of total run); 7-fold increase in MS sensitivity and 2-fold greater efficiency (50% peak width reduction) were observed when comparing the proposed method with the reference-quality HPLC-MS/MS system, whose assay performance has been previously documented. The UHPLC-MS/MS method led to an overall improvement in the limits of detection (LOD) and quantification (LOQ) for all the phenolic compounds studied. The recoveries ranged between 68% and 100% in urine and 61% and 100% in plasma. The accuracy; intra- and interday precision; and stability met with the acceptance criteria of the AOAC International norms. Due to the improvements in the analytical method; the total phenolic metabolites detected in plasma and urine in the pilot intervention study were 3 times higher than those detected by HPLC-MS/MS. Comparing with traditional methods; which require longer time of analysis; the methodology described is suitable for the analysis of phenolic compounds in a large number of plasma and urine samples in a reduced time frame.

Discovery of human urinary biomarkers of aronia-citrus juice intake by HPLC-q-TOF-based metabolomic approach.

Metabolomics has emerged in the field of food and nutrition sciences as a powerful tool for doing profiling approaches. In this context, HPLC-q-TOF-based metabolomics approach was applied to unveil changes in the urinary metabolome in human subjects (n = 51, 23 men and 28 women) after regular aronia-citrus juice (AC-juice) intake (250 mL/day) during 16 weeks compared to individuals given a placebo beverage. Samples were analyzed by HPLC-q-TOF followed by multivariate data analysis (orthogonal signal filtering-partial least square discriminant analysis) that discriminated relevant mass features related to AC-juice intake. The results showed that biomarkers of AC-juice intake including metabolites coming from metabolism of food components as proline betaine, ferulic acid, and two unknown mercapturate derivatives were identified. Discovery of new biomarkers of food intake will help in the building up of the food metabolome and facilitate future insights into the mechanisms of action of dietary components in population health.

Urinary isoxanthohumol is a specific and accurate biomarker of beer consumption.

Biomarkers of food consumption are a powerful tool to obtain more objective measurements of dietary exposure and to monitor compliance in clinical trials. In this study, we evaluated the effectiveness of urinary isoxanthohumol (IX) excretion as an accurate biomarker of beer consumption. A dose-response clinical trial, a randomized, crossover clinical trial, and a cohort study were performed. In the dose-response trial, 41 young volunteers (males and females, aged 28 ± 3 y) consumed different doses of beer at night and a spot urine sample was collected the following morning. In the clinical trial, 33 males with high cardiovascular risk (aged 61 ± 7 y) randomly were administered 30 g of ethanol/d as gin or beer, or an equivalent amount of polyphenols as nonalcoholic beer for 4 wk. Additionally, a subsample of 46 volunteers from the PREDIMED (Prevenciόn con Dieta Mediterránea) study (males and females, aged 63 ± 5 y) was also evaluated. Prenylflavonoids were quantified in urine samples by liquid chromatography coupled to mass spectrometry. IX urinary recovery increased linearly with the size of the beer dose in male volunteers. A significant increase in IX excretion (4.0 ± 1.6 µg/g creatinine) was found after consumption of beer and nonalcoholic beer for 4 wk (P < 0.001). Receiver operating characteristic curves showed that IX is able to discriminate between beer consumers and abstainers with a sensitivity of 67% and specificity of 100% (positive predictive value = 70%, negative predictive value = 100% in real-life conditions). IX in urine samples was found to be a specific and accurate biomarker of beer consumption and may be a powerful tool in epidemiologic studies.

Analytical condition setting a crucial step in the quantification of unstable polyphenols in acidic conditions: analyzing prenylflavanoids in biological samples by liquid chromatography-electrospray ionization triple quadruple mass spectrometry.

The interest in studying hops and beer prenylflavanoids, isoxanthohumol, xanthohumol, and 8-prenylnaringenin, has increased in recent years due to their biological activity as strong phytoestrogens and potent cancer chemopreventive agents. However, prenylflavanoids behave differently from most polyphenols, since they are unstable at acidic pH. To our knowledge, no published studies to date have considered the degradation of these compounds during analytical processes. In the present work, a new sensitive and specific method based on solid phase extraction and liquid chromatography coupled to electrospray ionization triple quadruple mass spectrometry (LC-ESI-MS/MS) was developed and validated. The new method was optimized to avoid degradation of the selected analytes, isoxanthohumol, xanthohumol, and 8-prenylnaringenin, throughout the analytical process and to reduce the urine matrix effect in LC-ESI-MS/MS assays. It was concluded that a neutral pH (pH 7.0) is necessary for the analysis of prenylflavanoids, in order to maintain the stability of compounds for at least 24 h. The addition of ascorbic acid to the media improved stability, calibration curves, coefficients of correlation, accuracy, and precision parameters. Mix-mode cation exchange sorbent yielded the best matrix effect factors and recoveries. Method validation results showed appropriate intraday and interday accuracy and precision (<15%). Recovery of isoxanthohumol, xanthohumol, and 8-prenylnaringenin was 97.1% ± 0.03, 105.8% ± 0.05, and 105.4% ± 0.04, respectively, and matrix effect factors were nearly 100%. The stability assay showed that analytes were stable for at least 24 h. The method was applied to quantify 10 human samples of urine and was able to quantify prenylflavanoids in urine after the consumption of a single dose of beer (330 mL).

Urinary metabolomics of phenolic compounds reveals biomarkers of type-2 diabetes within the PREDIMED trial.

BACKGROUND: Phenolic compounds have been associated with protective effects against type-2 diabetes (T2D). We used a metabolomics approach to determine urinary phenolic metabolites associated with T2D and fasting plasma glucose. METHODS: This case-control study within the PREDIMED trial included 200 participants at high cardiovascular risk, 102 of whom were diagnosed with T2D. A panel of urinary phenolic compounds were analysed using a novel method based on liquid chromatography coupled to mass spectrometry. Multivariate statistics and adjusted logistic regressions were applied to determine the most discriminant compounds and their association with T2D. The relationship between the discriminant phenolic compounds and plasma glucose was assessed using multivariable linear regressions. RESULTS: A total of 41 phenolic compounds were modeled in the orthogonal projection to latent structures discriminant analysis, and after applying adjusted logistic regressions two were selected as discriminant: dihydrocaffeic acid (OR = 0.22 (CI 95 %: 0.09; 0.52) per 1-SD, p-value = 0.021) and genistein diglucuronide (OR = 0.72 (CI 95%: 0.59; 0.88) per 1-SD, p-value = 0.021). Both metabolites were associated with a lower risk of suffering from T2D, but only dihydrocaffeic acid was inversely associated with plasma glucose (ß = -17.12 (95 % CI: -29.92; -4.32) mg/dL per 1-SD, p-value = 0.009). CONCLUSIONS: A novel method using a metabolomics approach was developed to analyse a panel of urinary phenolic compounds for potential associations with T2D, and two metabolites, dihydrocaffeic acid and genistein diglucuronide, were found to be associated with a lower risk of this condition.

Identification and Quantification of Urinary Microbial Phenolic Metabolites by HPLC-ESI-LTQ-Orbitrap-HRMS and Their Relationship with Dietary Polyphenols in Adolescents.

This study aimed to develop and validate a liquid chromatography/electrospray ionization-linear ion trap quadrupole-Orbitrap-high-resolution mass spectrometry (HPLC/ESI-LTQ-Orbitrap-HRMS) method to identify and quantify urinary microbial phenolic metabolites (MPM), as well as to explore the relationship between MPM and dietary (poly)phenols in Spanish adolescents. A total of 601 spot urine samples of adolescents aged 12.02 ± 0.41 years were analyzed. The quantitative method was validated for linearity, limit of detection, limit of quantification, recovery, intra- and inter-day accuracy and precision, as well as postpreparative stability according to the criteria established by the Association of Official Agricultural Chemists International. A total of 17 aglycones and 37 phase II MPM were identified and quantified in 601 spot urine samples. Phenolic acids were the most abundant urinary MPM, whereas stilbenes, hydroxytyrosol, and enterodiol were the least abundant. Urinary hydroxycoumarin acids (urolithins) were positively correlated with flavonoid and total (poly)phenol intake. An HPLC-ESI-LTQ-Orbitrap-HRMS method was developed and fully validated to quantify MPM. The new method was performed accurately and is suitable for MPM quantification in large epidemiological studies. Urinary lignans and urolithins are proposed as potential biomarkers of grain and nut intake in an adolescent population.

Metabolomics unveils urinary changes in subjects with metabolic syndrome following 12-week nut consumption.

Through an HPLC-Q-TOF-MS-driven nontargeted metabolomics approach, we aimed to discriminate changes in the urinary metabolome of subjects with metabolic syndrome (MetS), following 12 weeks of mixed nuts consumption (30 g/day), compared to sex- and age-matched individuals given a control diet. The urinary metabolome corresponding to the nut-enriched diet clearly clustered in a distinct group, and the multivariate data analysis discriminated relevant mass features in this separation. Metabolites corresponding to the discriminating ions (MS features) were then subjected to multiple tandem mass spectrometry experiments using LC-ITD-FT-MS, to confirm their putative identification. The metabolomics approach revealed 20 potential markers of nut intake, including fatty acid conjugated metabolites, phase II and microbial-derived phenolic metabolites, and serotonin metabolites. An increased excretion of serotonin metabolites was associated for the first time with nut consumption. Additionally, the detection of urinary markers of gut microbial and phase II metabolism of nut polyphenols confirmed the understanding of their bioavailability and bioactivity as a priority area of research in the determination of the health effects derived from nut consumption. The results confirmed how a nontargeted metabolomics strategy may help to access unexplored metabolic pathways impacted by diet, thereby raising prospects for new intervention targets.

Screening of the polyphenol content of tomato-based products through accurate-mass spectrometry (HPLC-ESI-QTOF).

Tomatoes, the second most important vegetable crop worldwide, are a key component in the so-called "Mediterranean diet" and its consumption has greatly increased worldwide over the past 2 decades, mostly due to a growing demand for tomato-based products such as ketchups, gazpachos and tomato juices. In this work, tomato-based products were analysed after a suitable work-up extraction procedure using liquid chromatography/electrospray ionisation-time of flight-mass spectrometry (HPLC-ESI-QTOF) with negative ion detection using information-dependent acquisition (IDA) to determine their phenolic composition. The compounds were confirmed by accurate mass measurements in MS and MS(2) modes. The elemental composition was selected according to the accurate masses and isotopic pattern. In this way, 47 compounds (simple phenolic and hydroxycinnamoylquinic acids and flavone, flavonol, flavanone and dihydrochalcone derivatives) were identified in tomato-based products, five of them, as far as was known, were previously unreported in tomatoes. The phenolic fingerprint showed that tomato-based products differ in phenolic composition, principally in protocatechuic acid-O-hexoside, apigenin and its glycosylated forms, quercetin-O-dihexoside, kaempferol-C-hexoside and eriodictyol-O-dihexoside. Gazpacho showed the highest number of phenolic compounds due to the vegetables added for its production.

Total Analysis of the Major Secoiridoids in Extra Virgin Olive Oil: Validation of an UHPLC-ESI-MS/MS Method.

Extra virgin olive oil (EVOO), one of the key foods of the Mediterranean diet, is distinguished by its high content of nutritional and antioxidant compounds compared to other vegetable oils. During EVOO production, the major secoiridoids of EVOO, oleacein, oleocanthal, ligstroside, and oleuropein aglycones, undergo a series of transformations to open- and closed-structure forms. The resulting mixture of compounds can become more complex during the analytical procedure, due to the keto-enol tautomerism of the open forms and their interaction with polar solvents, and therefore more challenging to analyze. Employing the same extraction method used to analyze the other EVOO phenolic compounds, we report here a simple UHPLC-ESI-MS/MS procedure for the quantification of those secoiridoids that is able to co-elute the different isomers of each compound. The method was validated following AOAC guidelines, and the matrix effect and recoveries were within satisfactory limits.

Metabolomics Technologies for the Identification and Quantification of Dietary Phenolic Compound Metabolites: An Overview.

In the search for natural products with properties that may protect against or slow down chronic and degenerative diseases (e.g., cancer, and cardiovascular and neurodegenerative conditions), phenolic compounds (PC) with benefits for human health have been identified. The biological effects of PC in vivo depend on their bioavailability, intestinal absorption, metabolism, and interaction with target tissues. The identification of phenolic compounds metabolites (PCM), in biological samples, after food ingestion rich in PC is a first step to understand the overall effect on human health. However, their wide range of physicochemical properties, levels of abundance, and lack of reference standards, renders its identification and quantification a challenging task for existing analytical platforms. The most frequent approaches to metabolomics analysis combine mass spectrometry and NMR, parallel technologies that provide an overview of the metabolome and high-power compound elucidation. In this scenario, the aim of this review is to summarize the pre-analytical separation processes for plasma and urine samples and the technologies applied in quantitative and qualitative analysis of PCM. Additionally, a comparison of targeted and non-targeted approaches is presented, not available in previous reviews, which may be useful for future metabolomics studies of PCM.

Tissue Distribution of Oleocanthal and Its Metabolites after Oral Ingestion in Rats.

Claims for the potential health benefits of oleocanthal (OLC), a dietary phenolic compound found in olive oil, are based mainly on in vitro studies. Little is known about the tissue availability of OLC, which is rapidly metabolized after ingestion. In this study, the distribution of OLC and its metabolites in rat plasma and tissues (stomach, intestine, liver, kidney, spleen, lungs, heart, brain, thyroid and skin) at 1, 2 and 4.5 h after the acute intake of a refined olive oil containing 0.3 mg/mL of OLC was examined by LC-ESI-LTQ-Orbitrap-MS. OLC was only detected in the stomach and intestine samples. Moreover, at 2 and 4.5 h, the concentration in the stomach decreased by 36% and 74%, respectively, and in the intestine by 16% and 33%, respectively. Ten OLC metabolites arising from phase I and phase II reactions were identified. The metabolites were widely distributed in rat tissues, and the most important metabolizing organs were the small intestine and liver. The two main circulating metabolites were the conjugates OLC + OH + CH3 and OLC + H2O + glucuronic acid, which may significantly contribute to the beneficial health effects associated with the regular consumption of extra virgin olive oil. However, more studies are necessary to determine the concentrations and molecular structures of OLC metabolites in human plasma and tissues when consumed with the presence of other phenolic compunds present in EVOO.

Absorption and metabolization of cytoprotective epicatechin thio conjugates in rats.

The epicatechin (EC) thio derivatives 4ß-(S-cysteinyl)-epicatechin (Cys-EC) and 4ß-(S-cysteaminyl)-epicatechin (Cya-EC) are compounds that may provide protection from oxidation via mechanisms involving either the flavonoid moiety or the nonphenolic cysteine or cysteamine part of the molecule. Because the metabolically modified molecules may be the actual active species, we estimated the absorption/metabolization of the thio derivatives through the small intestine in vitro and studied the body distribution of the compounds and their metabolites in rats. The analysis of the samples generated was done using a high-performance liquid chromatograph coupled to a UV detector and a tandem mass spectrometer. We show that Cya-EC follows the same phase II metabolization pattern as EC, whereas Cys-EC is transported with the intact catechol moiety through the small intestine and effectively metabolized systemically. We also found that Cya-EC generates Cys-EC in vivo, which provides evidence for a Cya-EC-mediated cytoprotective effect through cysteamine/cystine exchange with subsequent cysteine transport, ubiquitously throughout the organism.