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 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.

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.

Inhibition of Tryptophan Hydroxylases and Monoamine Oxidase-A by the Proton Pump Inhibitor, Omeprazole-In Vitro and In Vivo Investigations.

Serotonin (5-HT) is a hormone and neurotransmitter that modulates neural activity as well as a wide range of other physiological processes including cardiovascular function, bowel motility, and platelet aggregation. 5-HT synthesis is catalyzed by tryptophan hydroxylase (TPH) which exists as two distinct isoforms; TPH1 and TPH2, which are responsible for peripheral and central 5-HT, respectively. Due to the implication of 5-HT in a number of pathologies, including depression, anxiety, autism, sexual dysfunction, irritable bowel syndrome, inflammatory bowel disease, and carcinoid syndrome, there has been a growing interest in finding modulators of these enzymes in recent years. We thus performed high-throughput screening (HTS) using a fluorescence-based thermal shift assay (DSF) to search the Prestwick Chemical Library containing 1,280 compounds, mostly FDA-approved drugs, for TPH1 binders. We here report the identification of omeprazole, a proton pump inhibitor, as an inhibitor of TPH1 and TPH2 with low micromolar potency and high selectivity over the other aromatic amino acid hydroxylases. The S-enantiomer of omeprazole, esomeprazole, has recently also been described as an inhibitor of monoamine oxidase-A (MAO-A), the main enzyme responsible for 5-HT degradation, albeit with lower potency compared to the effect on TPH1 and TPH2. In order to investigate the net effect of simultaneous inhibition of TPH and MAO-A in vivo, we administered high-dose (100 mg/kg) omeprazole to CD-1 mice for 4 days, after which the animals were subjected to the tail suspension test. Finally, central (whole brain) and peripheral (serum) 5-HT content was measured using liquid chromatography-mass spectrometry (LC-MS). Omeprazole treatment significantly increased 5-HT concentrations, both in brain and in serum, and reduced the time spent immobile in the tail suspension test relative to vehicle control. Thus, the MAO-A inhibition afforded by high-dose omeprazole appears to overcome the opposing effect on 5-HT produced by inhibition of TPH1 and TPH2. Further modification of proton pump inhibitor scaffolds may yield more selective modulators of 5-HT metabolism.

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.

Quantitative Dietary Fingerprinting (QDF)-A Novel Tool for Comprehensive Dietary Assessment Based on Urinary Nutrimetabolomics.

Accurate dietary assessment is a challenge in nutritional research, needing powerful and robust tools for reliable measurement of food intake biomarkers. In this work, we have developed a novel quantitative dietary fingerprinting (QDF) approach, which enables for the first time the simultaneous quantitation of about 350 urinary food-derived metabolites, including (poly)phenolic aglycones, phase II metabolites, and microbial-transformed compounds, as well as other compounds (e.g., glucosinolates, amino acid derivatives, methylxanthines, alkaloids, and markers of alcohol and tobacco consumption). This method was fully validated for 220 metabolites, yielding good linearity, high sensitivity and precision, accurate recovery rates, and negligible matrix effects. Furthermore, 127 additional phase II metabolites were also included in this method after identification in urines collected from acute dietary interventions with various foods. Thus, this metabolomic approach represents one-step further toward precision nutrition and the objective of improving the accurateness and comprehensiveness in the assessment of dietary patterns and lifestyles.

Ex-Vivo and In-Vivo Assessment of Cyclamen europaeum Extract After Nasal Administration.

Rhinosinusitis is a prevalent disorder with a severe impact on the health-related quality of life. Saponins of Cyclamen europaeum exert a clinically proven curative effect on rhinosinusitis symptoms when instilled into the nasal cavity, however, more extensive preclinical assessment is required to better characterize the efficacy of this botanical extract. This work evaluates the potential use of a natural freeze-dried extract of C. europaeum given as topical nasal administration. Permeation experiment on porcine nasal mucosa was performed with Franz diffusion cells. Experiments in rabbits were performed to test for any toxicological, hematological, biochemical or histological evidence of systemic action. No theoretical levels of saponins were found in the receptor chamber of Franz diffusion cells. Hematological data did not show significant differences between control and experimental animals (p > 0.05). Histological studies also showed that enhanced secretory activity in response to intranasal administration was not accompanied by any visible signs of injury. An examination of the brain, lungs, liver, kidneys, spleen, and gastrointestinal organs did not reveal any abnormality. The absence of mucosal permeation of saponins and negligible probability of C. europaeum saponins absorption in the course of a therapeutic application was demonstrated.

Phytohormone Profiling Method for Rice: Effects of GA20ox Mutation on the Gibberellin Content of Japonica Rice Varieties.

Gibberellins (GAs) are a very important group of phytohormones involved in seed germination, vegetative growth, flowering, and fruit development, being only 4 of the 136 known bioactives: GA1, GA3, GA4, and GA7. It has been evidenced that mutations in the OsGA20ox-2 gene produce rice (Oryza sativa) dwarf varieties, which were one of the main pillars of the green revolution. In this work two main objectives were proposed: (i) develop a rapid and broad phytohormone profiling method and (ii) to study the effects on the GA content of the GA20ox-2 mutation in several rice developmental stages using three varieties (tall variety, elite variety, mutated variety). A phytohormone extraction using an SPE step and HPLC-MS/MS detection using a QqQ instrument was determined which resulted in limits of detection (LOD) and limits of quantification (LOQ) for GAs that varied between 0.1-0.7 and 0.3-2.3 pg ⋅ g-1 (f.w.) of rice sample, respectively, allowing highly sensitive phytohormones detection in samples. Moreover, a good reproducibility was obtained for the GAs as relative standard deviations (RSD) for a 40 ng ⋅ mL-1 pattern varied between 0.3 and 0.9%. Notoriously, GA1 was absent in the coleoptile and GA4 was the GA with higher content in the majority of developmental stages. We also observed a large content increase of the four bioactive GAs in the internode of the flag leaf of the mutated variety allowing to reach same height as the elite variety. Therefore, we provide a rapid and broad phytohormonal profiling method and evidence that the GA20ox-2 mutation is not the only factor generating dwarf varieties. To our knowledge, this is the first study that it has been reported such a high number of simultaneously analyzed gibberellins in rice samples (Oryza sativa ssp. japonica) in different tissues of different growth stages.

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.

Metabotypes of response to bariatric surgery independent of the magnitude of weight loss.

OBJECTIVE: Bariatric surgery is considered the most efficient treatment for morbid obesity and its related diseases. However, its role as a metabolic modifier is not well understood. We aimed to determine biosignatures of response to bariatric surgery and elucidate short-term metabolic adaptations. METHODS: We used a LC- and FIA-ESI-MS/MS approach to quantify acylcarnitines, (lyso)phosphatidylcholines, sphingomyelins, amino acids, biogenic amines and hexoses in serum samples of subjects with morbid obesity (n = 39) before and 1, 3 and 6 months after bariatric surgery. K-means cluster analysis allowed to distinguish metabotypes of response to bariatric surgery. RESULTS: For the first time, global metabolic changes following bariatric surgery independent of the baseline health status of the subjects have been revealed. We identify two metabolic phenotypes (metabotypes) at the interval 6 months-baseline after surgery, which presented differences in the levels of compounds of urea metabolism, gluconeogenic precursors and (lyso)phospholipid particles. Clinically, metabotypes were different in terms of the degree of improvement in insulin resistance, cholesterol, low-density lipoproteins and uric acid independent of the magnitude of weight loss. CONCLUSIONS: This study opens new perspectives and new hypotheses on the metabolic benefits of bariatric surgery and understanding of the biology of obesity and its associated diseases.

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.

A discovery-driven approach to elucidate urinary metabolome changes after a regular and moderate consumption of beer and nonalcoholic beer in subjects at high cardiovascular risk.

SCOPE: The aim of this work was to study the urinary metabolomics changes of participants that consumed beer, nonalcoholic beer (na-beer), and gin. METHODS AND RESULTS: Thirty-three males at high cardiovascular risk between 55 and 75 years old participated in an open, randomized, crossover, controlled trial with three nutritional interventions consisting of beer, na-beer, and gin for 4 wk. Diet and physical activity was monitored throughout the study and compliance was assessed by measurement of urinary isoxanthohumol. Metabolomic analysis was performed in urine samples by LC coupled to an LTQ-Orbitrap mass spectrometer combined with univariate and multivariate statistical analysis. Ten metabolites were identified. Eight were exogenous metabolites related to beer, na-beer, or gin consumption, but two of them were related to endogenic changes: hydroxyadipic acid linked to fatty acid oxidation, and 4-guanidinobutanoic acid, which correlated with a decrease in urinary creatinine. Plasmatic acylcarnitines were quantified by targeted MS. A regular and moderate consumption of beer and na-beer decreased stearoylcarnitine concentrations. CONCLUSION: Humulinone and 2,3-dihydroxy-3-methylvaleric acid showed to be potential biomarkers of beer and na-beer consumption. Moreover, the results of this trial provide new evidence that the nonalcoholic fraction of beer may increase fatty oxidation.

Bioavailability of tomato polyphenols is enhanced by processing and fat addition: Evidence from a randomized feeding trial.

SCOPE: Tomato contains a variety of phenolics associated with health-promoting properties. However, the effects of processing and the addition of oil during tomato sauce preparation on microbial metabolism of phenolics in the small intestine are still unclear. METHODS AND RESULTS: An open, controlled, randomized, and crossover feeding trial with 40 healthy volunteers was carried out to analyze the metabolites in plasma and urine after the consumption of tomato and tomato sauces, with tomato sauce enriched with refined olive oil (ROOE) and without refined olive oil (oil-free: OF). Ten phenolics in plasma and 93 metabolites in urine were quantified. Processing tomatoes into sauce enhanced the bioavailability of flavanones, flavanols, and some hydroxycinnamic acids, as reflected by the increase in the area under the plasma concentration versus time curve. An increase in their plasma half-life was also observed, particularly after ingestion of ROOE, possibly favored by enterohepatic circulation. A wide variety of gut microbial metabolites was also detected, namely flavanones, hydroxycinnamic acids, flavonols, hydroxyphenylpropanoic acids, hydroxyphenylacetic acids, and hydroxybenzoic acids. CONCLUSIONS: Flavanones and flavonols in ROOE presented higher bioavailability, suggesting that the processing undergone by the raw tomato improved their absorption.

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.

Alteration of cellular lipids and lipid metabolism markers in RTL-W1 cells exposed to model endocrine disrupters.

This work investigates the suitability of the rainbow trout liver cell line (RTL-W1) as an in-vitro model to study the ability of model endocrine disrupters, namely TBT, TPT, 4-NP, BPA and DEHP, to act as metabolic disrupters by altering cellular lipids and markers of lipid metabolism. Among the tested compounds, BPA and DEHP significantly increased the intracellular accumulation of triacylglycerols (TAGs), while all the compounds -apart from TPT-, altered membrane lipids - phosphatidylcholines (PCs) and plasmalogen PCs - indicating a strong interaction of the toxicants with cell membranes and cell signaling. RTL-W1 expressed a number of genes involved in lipid metabolism that were modulated by exposure to BPA, TBT and TPT (up-regulation of FATP1 and FAS) and 4-NP and DEHP (down-regulation of FAS and LPL). Multiple and complex modes of action of these chemicals were observed in RTL-W1 cells, both in terms of expression of genes related to lipid metabolism and alteration of cellular lipids. Although further characterization is needed, this might be a useful model for the detection of chemicals leading to steatosis or other diseases associated with lipid metabolism in fish.