Comparative metabolomic study of transgenic versus conventional soybean using capillary electrophoresis–time-of-flight mass spectrometry

In this work, capillary electrophoresis–time-of-flight mass spectrometry (CE–TOF-MS) is proposed to identify and quantify the main metabolites found in transgenic soybean and its corresponding non-transgenic parental line both grown under identical conditions. The procedure includes optimization of metabolites extraction, separation by CE, on-line electrospray-TOF-MS analysis and data evaluation. A large number of extraction procedures and background electrolytes are tested in order to obtain a highly reproducible and sensitive analytical methodology. Using this approach, a large number of metabolites were tentatively identified based on the high mass accuracy provided by TOF-MS analyzer, together with the isotopic pattern and expected electrophoretic mobility of these compounds. In general, the same metabolites and in similar amounts were found in the conventional and transgenic variety. However, significant differences were also observed in some specific cases when the conventional variety was compared with its corresponding transgenic line. The selection of these metabolites as possible biomarkers of transgenic soybean is discussed, although a larger number of samples need to be analyzed in order to validate this point. It is concluded that metabolomic procedures based on CE-MS can open new perspectives in the study of transgenic foods in order to corroborate (or not) the equivalence with their conventional counterparts.Peer reviewe

Targeted and Untargeted Metabolomics to Explore the Bioavailability of the Secoiridoids from a Seed/Fruit Extract (Fraxinus angustifolia Vahl) in Human Healthy Volunteers: A Preliminary Study

The bark, seeds, fruits and leaves of the genus Fraxinus (Oleaceae) which contain a wide range of phytochemicals, mostly secoiridoid glucosides, have been widely used in folk medicine against a number of ailments, yet little is known about the metabolism and uptake of the major Fraxinus components. The aim of this work was to advance in the knowledge on the bioavailability of the secoiridoids present in a Fraxinus angustifolia Vahl seed/fruit extract using both targeted and untargeted metabolomic analyses. Plasma and urine samples from nine healthy volunteers were taken at specific time intervals following the intake of the extract and analyzed by UPLC-ESI-QTOF. Predicted metabolites such as tyrosol and ligstroside-aglycone glucuronides and sulfates were detected at low intensity. These compounds reached peak plasma levels 2 h after the intake and exhibited high variability among the participants. The ligstroside-aglycone conjugates may be considered as potential biomarkers of the Fraxinus secoiridoids intake. Using the untargeted approach we additionally detected phenolic conjugates identified as ferulic acid and caffeic acid sulfates, as well as hydroxybenzyl and hydroxyphenylacetaldehyde sulfate derivatives which support further metabolism of the secoiridoids by phase I and (or) microbial enzymes. Overall, the results of this study suggest low uptake of intact secoiridoids from a Fraxinus angustifolia Vahl extract in healthy human volunteers and metabolic conversion by esterases, glycosidases, and phase II sulfo- and glucuronosyl transferases to form smaller conjugated derivatives.This work was financially supported by the Centre for the Development of Industrial Technology (CDTI) from the Spanish Government (NEMAF project) and R.G.V., P.F-B. and M-T.G.C. are participating to the COST Action FA1403 Positive (Interindividual variation in response to consumption of plant food bioactives and determinants involved). We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI).Peer reviewe

The Breast Cancer Resistance Protein (BCRP/ABCG2) influences the levels of enterolignans and their metabolites in plasma, milk and mammary gland

P. 648-654Lignans are phytoestrogens widely used in dietary supplements and functional foods. After oral ingestion, these polyphenols are metabolized to enterolignans, the main gut microbiota-derived metabolites with weak estrogenic/anti-estrogenic activities. The ABCG2 transporter is highly expressed in the mammary gland and could be responsible for enterolignan accumulation. We aimed here at evaluating the levels of enterolignans and their conjugates in plasma, milk and mammary tissue from wild-type and knockout Abcg2-/- female mice after a lignan-enriched diet for one week. In vitro transepithelial transport of enterolignans was also assayed with ABCG2- transduced cells. Enterolactone and enterodiol levels were higher in plasma and lower in milk from Abcg2-/- compared with wild-type mice. Both enterolactone and enterodiol were accumulated in the mammary gland but with significant differences only for enterolactone. Our results suggest that ABCG2 may be determinant for plasma and milk levels of enterolignans whose accumulation could exert chemopreventive effects against breast cancerS

Altered tissue distribution of flaxseed lignans and their metabolites in Abcg2 knockout mice

Lignans are dietary polyphenols, which are metabolized by gut microbiota into the phytoestrogenic metabolites enterolignans, mainly enterolactone and enterodiol. Breast Cancer Resistance Protein (BCRP/ABCG2) is an efflux transporter that affects the plasma and milk secretion of several drugs and natural compounds. We hypothesized here that Abcg2 could influence the levels of lignans and their derived metabolites in target tissues. Consequently, we aimed to evaluate the role of Abcg2 in the tissue distribution of these compounds. We used Abcg2−/− knockout and wild-type male mice fed with a lignan-enriched diet for one week and analysed their plasma, small intestine, colon, liver, kidneys and testicles. High levels of lignans as well as enterolignans and their glucuronide and sulfate conjugates in the small intestine and colon were detected, with higher concentrations of the conjugates in the wildtype compared with Abcg2−/− mice. Particularly relevant was the detection of 24-fold and 8-fold higher concentrations of enterolactone-sulfate and enterolactone-glucuronide, respectively, in the kidney of Abcg2−/− compared with wild-type mice. In conclusion, our study showed that lignans and their derived metabolites were in vivo substrates of Abcg2, which affected their plasma and tissue levels. These results highlight the role of Abcg2 in influencing the health-beneficial properties of dietary lignans.S

Nano- and rapid resolution liquid chromatography-electrospray ionization-time of flight mass spectrometry to identify and quantify phenolic compounds in olive oil

The applicability of nano-liquid chromatography coupled to electrospray ionization-time of flight-mass spectrometry (nanoLC-ESI-TOF MS) for the analysis of phenolic compounds in olive oil was studied and compared with a HPLC method. After the injection, the compounds were focused on a short capillary trapping column (100 μm i.d., effective length 20 mm, 5 μm particle size) and then nanoLC analysis was carried out in a fused silica capillary column (75 μm i.d., effective length 10 cm, 3 μm particle size) packed with C18 stationary phase. The mobile phase was a mixture of water + 0.5% acetic acid and acetonitrile eluting at 300 nL/min in a gradient mode. Phenolic compounds from different families were identified and quantified. The quality parameters of the nanoLC method (linearity, limits of detection and quantification, repeatability) were evaluated and compare to those obtained with HPLC. The new methodology presents better sensitivity (reaching LOD values below 1 ppb) with less consumption of mobile phases, but worse repeatability, especially inter-day repeatability, doing more difficult to get highly accurate quantification. The results described in this paper open up the application fields of this technique to cover a larger variety of compounds and its advantages will make it especially useful for the analysis of samples containing low concentration of phenolic compounds, as for instance, in biological samples.Ministry of Education and Science (FPU, AP2005- 4356 and Proyect AGL 2008-05108-CO3-03/ALI), and Junta de Andalucía (Proyect P07-AGR-02619)

Uptake and metabolism of olive oil polyphenols in human breast cancer cells using nano-liquid chromatography coupled to electrospray ionization–time of flight-mass spectrometry

Polyphenols from extra virgin olive oil (EVOO), a main component of the Mediterranean diet, have demonstrated repeatedly anti-tumor activity in several in vitro and in vivo studies. However, little is known about the efficiency of the absorption process and metabolic conversion of these compounds at cellular level. In this study, a nano liquid chromatography–electrospray ionization–time of flight mass spectrometry (nanoLC–ESI–TOF MS) method was developed to study the cellular uptake and metabolism of olive oil phenols in JIMT-1 human breast cancer cells. After incubation for different time periods with EVOO-derived phenolic extracts, culture media, cytosolic fraction and solid particles fraction were separated and analyzed. Most of the free phenols, mainly hydroxytyrosol, its secoiridoid derivatives, and the flavonoid luteolin, disappeared in the culture media in different ways and at different times. Besides, several metabolites were detected in the culture media, fact that may indicate absorption and intracellular metabolism followed by rapid cellular export. Low intracellular accumulation was observed with only traces of some compounds detected in the cytosolic and solid particles fractions. Methylated conjugates were the major metabolites detected, suggesting a catalytic action of catechol-O-methyl transferase (COMT) in cancer cells.The authors are very grateful to Ministry of Education and Science (FPU, AP2005-4356) and Junta de Andalucia (project P09-FQM-5469, project P07-AGR-02619 and AGL 2008-05108- CO3-03/ALI). Besides, this work was supported in part by Instituto de Salud Carlos III (Ministerio de Sanidad y Consumo, Fondo de Investigación Sanitaria – FIS, Spain, Grants CP05-00090, PI06-0778 and RD06-0020-0028)

The Breast Cancer Resistance Protein (BCRP/ABCG2) as a key player in the tissue distribution of flaxseed lignans and their metabolites

25 p.Lignans are dietary polyphenols, which are metabolized by the gut microbiota into the phytoestrogenic metabolites enterolignans, mainly enterolactone and enterodiol. The Breast Cancer Resistance Protein (BCRP/ABCG2) is an efflux transporter that affects plasma and milk secretion of several drugs and natural compounds. We hypothesized here that Abcg2 could influence levels of lignans and their derived metabolites in target tissues. Consequently, we aimed to evaluate the role of Abcg2 in the tissue distribution of these compounds. We used Abcg2-/- knockout and wild-type male mice fed with a lignanenriched diet for one week and analysed plasma, small intestine, colon, liver, kidneys and testicles. High levels of lignans as well as enterolignans and their glucuronide and sulfate conjugates in the small intestine and colon were detected, with higher concentrations of the conjugates in the wild-type compared with Abcg2-/- mice. Particularly relevant was the detection of 24-fold and 8-fold higher concentrations of enterolactone-sulfate and enterolactone-glucuronide, respectively, in the kidney of Abcg2-/- compared with wild-type mice. In conclusion, our study showed that lignans and their derived metabolites were in vivo substrates of Abcg2, which affected their plasma and tissue levels. These results highlight the role of Abcg2 in influencing the health-beneficial properties of dietary lignan

Flaxseed-enriched diets change milk concentration of the antimicrobial danofloxacin in sheep

8 p.Flaxseed is the most common and rich dietary source of lignans and is an acceptable supply of energy for livestock. Flaxseed lignans are precursors of enterolignans, mainly enterolactone and enterodiol, produced by the rumen and intestinal microbiota of mammals and have many important biological properties as phytoestrogens. Potential food-drug interactions involving flaxseed may be relevant for veterinary therapy, and for the quality and safety of milk and dairy products. Our aim was to investigate a potential food-drug interaction involving flaxseed, to explore whether the inclusion of flaxseed in sheep diet affects concentration of the antimicrobial danofloxacin in milkS

Exploratory analysis of human urine by LC–ESI-TOF MS after high intake of olive oil: understanding the metabolism of polyphenols

Olive oil polyphenols have important biological properties which closely depend on their bioavailability, therefore it is essential to understand how polyphenols are absorbed, metabolized and eliminated from the body. An analytical methodology based on rapid resolution liquid chromatography (RRLC) coupled to mass spectrometry detection with a time of flight analyzer (RRLC-ESI-TOF MS) was developed for the determination of the main olive oil phenolic compounds and their metabolites in human urine. Urine samples from ten healthy volunteers were collected before and 2, 4 and 6 h after the intake of 50 mL of extra-virgin olive oil. The proposed method includes liquid-liquid extraction with ethyl acetate that provides extraction recoveries of the phenolic compounds studied between 35 and 75% from spiked urine samples. Good repeatability was obtained, since the relative standard deviations (RSDs) of peak areas in the intra- and inter-day studies were 4.3 and 6.5%, respectively. Statistical studies allowed us to discriminate between the urine samples before and after the intake, and facilitated to find out the m/z values responsible of this discrimination. Based on the very accurate mass information and the isotopic pattern provided by TOF-MS analyzer, together with other available information, ten of these biomarkers and more than 50 metabolites, obtained through phase I and phase II biotransformation reactions, were tentatively identified. Additionally, kinetic studies of the metabolites identified as possible biomarkers were developed, obtaining maximal values in the first two hours for most compounds.The authors are very grateful to Ministry of Education and Science (FPU, AP2005-4356 and Project AGL 2008- 05108-613 CO3-03/ALI), and Junta de Andalucía (P07-AGR-02629 and P09-FQM-5469)