Aida Serra

Bioavailability of procyanidin dimers and trimers and matrix food effects in in vitro and in vivo models

Among procyanidins (PC), monomers, such as catechin and epicatechin, have been widely studied, whereas dimer and trimer oligomers have received much less attention, despite their abundance in our diet. Recent studies have showed that as dimers and trimers could be important in determining the biological effects of procyanidin-rich food, understanding their bioavailability and metabolism is fundamental. The purpose of the present work is to study the stability of PC under digestion conditions, the metabolism and the bioavailability by using a combination of in vitro and in vivo models. Simultaneously, the matrix effect of a carbohydrate-rich food on the digestibility and bioavailability of PC is investigated. The results show a high level of stability of PC under gastric and duodenal digestion conditions. However, the pharmacokinetic study revealed limited absorption. Free forms of dimers and trimers have been detected in rat plasma, reaching the maximum concentration 1 h after oral intake of a grape seed extract.

Analysis of food polyphenols by ultra high-performance liquid chromatography coupled to mass spectrometry: an overview.

Phenolic compounds, which are widely distributed in plant-derived foods, recently attracted much attention due to their health benefits, so their determination in food samples is a topic of increasing interest. In the last few years, the development of chromatographic columns packed with sub-2μm particles and the modern high resolution mass spectrometry (MS) have opened up new possibilities for improving the analytical methods for complex sample matrices, such as ingredients, foods and biological samples. In addition, they have emerged as an ideal tool for profiling complex samples due to its speed, efficiency, sensitivity and selectivity. The present review addresses the use of the improved liquid chromatography (LC), ultra-high performance LC (UHPLC), coupled to MS or tandem MS (MS/MS) as the detector system for the determination of phenolic compounds in food samples. Additionally, the different strategies to extract, quantify the phenolic compounds and to reduce the matrix effect (%ME) are also reviewed. Finally, a briefly outline future trends of UHPLC-MS methods is commented.

Distribution of olive oil phenolic compounds in rat tissues after administration of a phenolic extract from olive cake

SCOPE The distribution and accumulation of olive oil phenolic compounds in the body are topics lacked of information. The aim of this study was to evaluate the bioavailability, metabolism and distribution of phenolic compounds from olive cake. METHODS AND RESULTS The metabolism and distribution of phenolic compounds were examined by UPLC-MS/MS after an acute intake of a phenolic extract from olive cake, analyzing plasma and tissues (heart, brain, liver, kidney, spleen, testicle and thymus) 1, 2 and 4 h after ingestion using Wistar rats as the in vivo model. The results showed a wide distribution of phenolic compounds and their metabolites in the tissues, with a main detoxification route through the kidneys. Highlighting the quantification of the free forms of some phenolic compounds, such as oleuropein derivative in plasma (Cmax 4 h: 24 nmol/L) and brain (Cmax 2 h: 2.8 nmol/g), luteolin in kidney (Cmax 1 h: 0.04 nmol/g), testicle (Cmax 2 h: 0.07 nmol/g) and heart (Cmax 1 h: 0.47 nmol/g); and hydroxytyrosol in plasma (Cmax 2 h: 5.2 nmol/L), kidney (Cmax 4 h: 3.8 nmol/g) and testicle (Cmax 2 h: 2.7 nmol/g). CONCLUSION After a single ingestion of olive oil phenolic compounds, these were absorbed, metabolized and distributed through the blood stream to practically all parts of the body, even across the blood-brain barrier.

Determination of procyanidins and their metabolites in plasma samples by improved liquid chromatography–tandem mass spectrometry

An off-line solid-phase extraction (SPE) and ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for determining procyanidins, catechin, epicatechin, dimer, and trimer in plasma samples. In the validation procedure of the analytical method, linearity, precision, accuracy, detection limits (LODs), quantification limits (LOQs), and the matrix effect were studied. Recoveries of the procyanidins were higher than 84%, except for the trimer, which was 65%. The LODs and LOQs were lower than 0.003 and 0.01 microM, respectively, for all the procyanidins studied, except for the trimers, which were 0.8 and 0.98 microM, respectively. This methodology was then applied for the analysis of rat plasma obtained 2h after ingestion of grape seed phenolic extract. Monomers (catechin and epicatechin), dimer and trimer in their native form were detected and quantified in plasma samples, and their concentration ranged from 0.85 to 8.55 microM. Moreover, several metabolites, such as catechin and epicatechin glucuronide, catechin and epicatechin methyl glucuronide, and catechin and epicatechin methyl-sulphate were identified. These conjugated forms were quantified, in reference to the respective unconjugated form, showing concentrations between 0.06 and 23.90 microM.

Rapid methods to determine procyanidins, anthocyanins, theobromine and caffeine in rat tissues by liquid chromatography-tandem mass spectrometry.

Rapid, selective and sensitive methods were developed and validated to determine procyanidins, anthocyanins and alkaloids in different biological tissues, such as liver, brain, the aorta vein and adipose tissue. For this purpose, standards of procyanidins (catechin, epicatechin, and dimer B(2)), anthocyanins (cyanidin-3-glucoside and malvidin-3-glucoside) and alkaloids (theobromine, caffeine and theophylline) were used. The methods included the extraction of homogenized tissues by off-line liquid-solid extraction, and then solid-phase extraction to analyze alkaloids, or microelution solid-phase extraction plate for the analysis of procyanidins and anthocyanins. The eluted extracts were then analyzed by ultra-performance liquid chromatography-electrospray ionization-tandem mass spectrometry, using a triple quadrupole as the analyzer. The optimum extraction solution was water/methanol/phosphoric acid 4% (94/4.5/1.5, v/v/v). The extraction recoveries were higher than 81% for all the studied compounds in all the tissues, except the anthocyanins, which were between 50 and 65% in the liver and brain. In order to show the applicability of the developed methods, different rat tissues were analyzed to determine the procyanidins, anthocyanins and alkaloids and their generated metabolites. The rats had previously consumed 1g of a grape pomace extract (to analyze procyanidins and anthocyanins) or a cocoa extract (to analyze alkaloids) per kilogram of body weight. Different tissues were extracted 4h after administration of the respective extracts. The analysis of the metabolites revealed a hepatic metabolism of procyanidins. The liver was the tissue which produced a greater accumulation of these metabolites.

Extracellular vesicles are rapidly purified from human plasma by PRotein Organic Solvent PRecipitation (PROSPR).

Extracellular vesicles (EVs) such as exosomes and microvesicles mediate intercellular communication and regulate a diverse range of crucial biological processes. Host cells that are damaged, infected or transformed release biomarker-containing EVs into the peripheral circulation, where they can be readily accessed for use in diagnostic or prognostic testing. However, current methods of EV isolation from blood plasma are complex and often require relatively large sample volumes, hence are inefficient for widespread use in clinical settings. Here, we report a novel and inexpensive method of rapidly isolating EVs from small volumes of human blood plasma by PRotein Organic Solvent PRecipitation (PROSPR). PROSPR encompasses a rapid three-step protocol to remove soluble proteins from plasma via precipitation in cold acetone, leaving the lipid-encapsulated EVs behind in suspension. This generates higher purity EVs that can then be obtained from filtration or classical ultracentrifugation methods. We foresee that PROSPR-based purification of EVs will significantly accelerate the discovery of new disease biomarkers and the characterization of EVs with potential for clinical applications.

Validation of determination of plasma metabolites derived from thyme bioactive compounds by improved liquid chromatography coupled to tandem mass spectrometry

In the present study, a selective and sensitive method, based on microelution solid-phase extraction (μSPE) plate and ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) was validated and applied to determine the plasma metabolites of the bioactive compounds of thyme. For validation process, standards of the more representative components of the phenolic and monoterpene fractions of thyme were spiked in plasma samples and then the quality parameters of the method were studied. Extraction recoveries (%R) of the studied compounds were higher than 75%, and the matrix effect (%ME) was lower than 18%. The LODs ranged from 1 to 65 μg/L, except for the thymol sulfate metabolite, which was 240 μg/L. This method was then applied for the analysis of rat plasma obtained at different times, from 0 to 6h, after an acute intake of thyme extract (5 g/kg body weight). Different thyme metabolites were identified and were mainly derived from rosmarinic acid (coumaric acid sulfate, caffeic acid sulfate, ferulic acid sulfate, hydroxyphenylpropionic acid sulfate, dihydroxyphenylpropionic acid sulfate and hydroxybenzoic acid) and thymol (thymol sulfate and thymol glucuronide). The most abundant thyme metabolites generated were hydroxyphenylpropionic acid sulfate and thymol sulfate, their respective concentrations in plasma being 446 and 8464 μM 1h after the intake of the thyme extract.

Gender differences in white matter pathology and mitochondrial dysfunction in Alzheimer’s disease with cerebrovascular disease

BackgroundDementia risk in women is higher than in men, but the molecular neuropathology of this gender difference remains poorly defined. In this study, we used unbiased, discovery-driven quantitative proteomics to assess the molecular basis of gender influences on risk of Alzheimer’s disease with cerebrovascular disease (AD + CVD).ResultsWe detected modulation of several redox proteins in the temporal lobe of AD + CVD subjects, and we observed sex-specific alterations in the white matter (WM) and mitochondria proteomes of female patients. Functional proteomic analysis of AD + CVD brain tissues revealed increased citrullination of arginine and deamidation of glutamine residues of myelin basic protein (MBP) in female which impaired degradation of degenerated MBP and resulted in accumulation of non-functional MBP in WM. Female patients also displayed down-regulation of ATP sub-units and cytochromes, suggesting increased severity of mitochondria impairment in women.ConclusionsOur study demonstrates that gender-linked modulation of white matter and mitochondria proteomes influences neuropathology of the temporal lobe in AD + CVD.

A high-throughput peptidomic strategy to decipher the molecular diversity of cyclic cysteine-rich peptides

Cyclotides are plant cyclic cysteine-rich peptides (CRPs). The cyclic nature is reported to be gene-determined with a precursor containing a cyclization-competent domain which contains an essential C-terminal Asn/Asp (Asx) processing signal recognized by a cyclase. Linear forms of cyclotides are rare and are likely uncyclizable because they lack this essential C-terminal Asx signal (uncyclotide). Here we show that in the cyclotide-producing plant Clitoria ternatea, both cyclic and acyclic products, collectively named cliotides, can be bioprocessed from the same cyclization-competent precursor. Using an improved peptidomic strategy coupled with the novel Asx-specific endopeptidase butelase 2 to linearize cliotides at a biosynthetic ligation site for transcriptomic analysis, we characterized 272 cliotides derived from 38 genes. Several types of post-translational modifications of the processed cyclotides were observed, including deamidation, oxidation, hydroxylation, dehydration, glycosylation, methylation, and truncation. Taken together, our results suggest that cyclotide biosynthesis involves ‘fuzzy’ processing of precursors into both cyclic and linear forms as well as post-translational modifications to achieve molecular diversity, which is a commonly found trait of natural product biosynthesis.

Flavanol metabolites distribute in visceral adipose depots after a long-term intake of grape seed proanthocyanidin extract in rats

A considerable number of epidemiological investigations and intervention studies have supported an association between the intake of flavanol- and proanthocyanidin-containing foods and a decreased risk of metabolic diseases. Nonetheless, less is know about the capacity of tissues to accumulate flavanols and/or their metabolites. The main objective of the present study was to determine (n 20) plasma bioavailability and disposition in the liver, muscle, brown adipose tissue (BAT) and white adipose tissues (mesenteric and perirenal) in rats after a long-term consumption of three doses of grape seed phenolic extract (5, 25 and 50 mg/kg body weight) for 21 d in order to determine whether there is a dose-response relationship. Glucuronidated conjugates (total glucuronidated conjugates: C(5 mg/kg) 1·9; C(25 mg/kg) 6·4; C(50 mg/kg) 27·7 μmol/l plasma) followed by methyl glucuronidated conjugates (total methyl glucuronidated conjugates: C(5 mg/kg) 1·98; C(25 mg/kg) 4·48; C(50 mg/kg) 12·5 μmol/l plasma) were the main flavanol metabolites quantified in plasma, also detecting a dimer in its free form (C(25 mg/kg) 0·74; C(50 mg/kg) 0·79 μmol/l plasma). Each of the studied organs has a particular behaviour of accumulation and response to the assayed grape seed extract doses, with an exponential bioavailability-dose relationship in BAT, in which flavanols could play an important role in the reduction or prevention of obesity, modulating the functionality of that tissue.