María José Motilva

Metabolites Involved in Oleuropein Accumulation and Degradation in Fruits of Olea europaea L.: Hojiblanca and Arbequina Varieties

The biosynthetic pathway of oleuropein (from 7-ketologanin, oleoside-11-methyl ester, 7-β-1-d-glucopyranosyl-11-methyl oleoside, and ligstroside to oleuropein) was investigated in two fruit species of Oleaceae, namely, Arbequina and Hojiblanca. Main oleuropein precursors and their metabolites, produced by the enzymatic hydrolysis mediated by β-glucosidase, were identified and quantified to establish the oleuropein transformation pathway. Changes in the concentration of these compounds were measured by direct control of in vivo fruit tissue during their ripening. High contents of aglycones at the initial stage of the process were caused by the high activity of β-glucosidase, which supports that oleuropein biosynthesis is coupled with enzymatic hydrolysis, producing its aglycone form. The low oleuropein content at this initial stage was caused by the imbalance between catabolic and anabolic pathways, favoring the former ones. Once the main polyphenol synthesis phase was completed, the biosynthetic capacity diminished and the content of all compounds decreased. Mass balance revealed that precursors of oleuropein, which are rapidly transformed by β-glucosidase and esterases, scarcely contributed to the accumulation of oleuropein. The biosynthetic pathway proposed by Damtoft applies for both varieties, but our study reveals that the β-glucosidase enzyme is involved in oleuropein synthesis. This enzyme shows high substrate specificity to oleuropein, which consequently is degraded to its aglycone form, with diminished efficacy of oleuropein biosynthesis. Different enzymatic activities of varieties will result in oleuropein accumulation and metabolic transformation of phenols.

Multicompartmental LC-Q-TOF-based metabonomics as an exploratory tool to identify novel pathways affected by polyphenol-rich diets in mice.

Metabonomics has recently been used to study the physiological response to a given nutritional intervention, but such studies have usually been restricted to changes in either plasma or urine. In the present study, we demonstrate that the use of LC-Q-TOF-based metabolome analyses (foodstuff, plasma, urine, and caecal content metabolomes) in mice offer higher order information, including intra- and intercompartment relationships. To illustrate this, we performed an intervention study with three different phenolic-rich extracts in mice over 3 weeks. Both unsupervised (PCA) and supervised (PLS-DA) multivariate analyses used for pattern recognition revealed marked effects of diet in each compartment (plasma, urine, and caecal contents). Specifically, dietary intake of phenolic-rich extract affects pathways such as bile acid and taurine metabolism. Q-TOF-based metabonomics demonstrated that the number of correlations is higher in caecal contents and urine than in plasma. Moreover, intercompartment correlations showed that caecal contents-plasma correlations are the most frequent in mice, followed by plasma-urine ones. The number of inter- and intracompartment correlations is significantly affected by diet. These analyses reveal the complexity of interorgan metabolic relationships and their sensitivity to dietary changes.

Complementary phenol-enriched olive oil improves HDL characteristics in hypercholesterolemic subjects. A randomized, double-blind, crossover, controlled trial. The VOHF study.

SCOPE Consumption of olive oil (OO) phenolic compounds (PCs) has beneficial effects on lipid profile. HDL functionality is currently considered to be a more important issue than its circulating quantity. Our aim was to assess whether functional virgin olive oils (FVOOs), one enriched with its own PC (500 ppm; FVOO) and another with OOPC (250 ppm) plus additional complementary PCs from thyme (250 ppm) (total: 500 ppm; FVOOT (functional virgin olive oil with thyme)), could improve HDL functionality related properties versus a virgin OO control (80 ppm; VOO). METHODS AND RESULTS In a randomized, double-blind, crossover, controlled trial, 33 hypercholesterolemic volunteers received 25 mL/day of VOO, FVOO, and FVOOT during 3 wk. HDL cholesterol increased 5.74% (p < 0.05) versus its baseline after the FVOOT consumption in the participants without hypolipidemic medication. We detected, after FVOOT consumption, an increase in HDL2 -subclass (34.45, SD = 6.38) versus VOO intake (32.73, SD = 6.71). An increment in esterified cholesterol/free cholesterol and phospholipids/free cholesterol in HDL was observed after FVOOT consumption (1.73, SD = 0.56; 5.44, SD = 1.39) compared with VOO intervention (1.53, SD = 0.35; 4.97, SD = 0.81) and FVOO intervention (1.50, SD = 0.33; 4.97, SD = 0.81). Accordingly, lecithin-cholesterol acyltransferase mass increased after FVOOT consumption (1228 μg/mL, SD = 130), compared with VOO consumption (1160 μg/mL, SD = 144). An improvement in HDL oxidative-status was reflected after FVOOT consumption versus its baseline, given an increment in the paraoxonase activity (118 × 10(3) U/L, SD = 24). CONCLUSION FVOOT improves HDL-subclass distribution and composition, and metabolism/antioxidant enzyme activities. FVOOT could be a useful dietary tool in the management of high cardiovascular risk patients.

β-Glucosidase involvement in the formation and transformation of oleuropein during the growth and development of olive fruits (Olea europaea L. cv. Arbequina) grown under different farming practices.

The present study investigates oleuropein metabolism, as well as the involvement of β-glucosidase during the growth, development, and ripening of olive fruit. The results show that in olive fruit the in vivo formation and transformation of oleuropein takes place in three different stages. The first one is characterized by a net accumulation of oleuropein and occurs in the immature fruit. In the second stage, associated with the green and light-green fruits, oleuropein content is maintained practically constant, and finally, a third stage that begins in the green-yellow fruit is characterized by a progressive decline of the oleuropein concentration. Our findings confirm that in the absence of β-glucosidase the Damtoft-proposed pathway is active and that net synthesis of oleuropein is unquestionable. β-Glucosidase activity plays a key role in the oleuropein metabolism catalyzing its in vivo hydrolysis.

Influence of Phenol-Enriched Olive Oils on Human Intestinal Immune Function.

Olive oil (OO) phenolic compounds (PC) are able to influence gut microbial populations and metabolic output. Our aim was to investigate whether these compounds and changes affect the mucosal immune system. In a randomized, controlled, double blind cross-over human trial, for three weeks, preceded by two-week washout periods, 10 hypercholesterolemic participants ingested 25 mL/day of three raw virgin OO differing in their PC concentration and origin: (1) an OO containing 80 mg PC/kg (VOO); (2) a PC-enriched OO containing 500 mg PC/kg from OO (FVOO); and (3) a PC-enriched OO containing a mixture of 500 mg PC/kg from OO and thyme (1:1, FVOOT). Intestinal immunity (fecal immunoglobulin A (IgA) and IgA-coated bacteria) and inflammation markers (C-reactive protein (CRP) and fecal interleukin 6 (IL-6), tumor necrosis factor α (TNFα) and calprotectin) was analyzed. The ingestion of high amounts of OO PC, as contained in FVOO, tended to increase the proportions of IgA-coated bacteria and increased plasma levels of CRP. However, lower amounts of OO PC (VOO) and the combination of two PC sources (FVOOT) did not show significant effects on the variables investigated. Results indicate a potential stimulation of the immune system with very high doses of OO PC, which should be further investigated.

Interplay between TDP-43 and docosahexaenoic acid-related processes in amyotrophic lateral sclerosis

BACKGROUND Docosahexaenoic acid (DHA), a key lipid in nervous system homeostasis, is depleted in the spinal cord of sporadic amyotrophic lateral sclerosis (sALS) patients. However, the basis for such loss was unknown. METHODS DHA synthetic machinery was evaluated in spinal cord samples from ALS patients and controls by immunohistochemistry and western blot. Further, lipid composition was measured in organotypic spinal cord cultures by gas chromatography and liquid chromatography coupled to mass spectrometry. In these samples, mitochondrial respiratory functions were measured by high resolution respirometry. Finally, Neuro2-A and stem cell-derived human neurons were used for evaluating mechanistic relationships between TDP-43 aggregation, oxidative stress and cellular changes in DHA-related proteins. RESULTS ALS is associated to changes in the spinal cord distribution of DHA synthesis enzymatic machinery comparing ten ALS cases and eight controls. We found increased levels of desaturases (ca 95% increase, p<0.001), but decreased amounts of DHA-related β-oxidation enzymes in ALS samples (40% decrease, p<0.05). Further, drebrin, a DHA-dependent synaptic protein, is depleted in spinal cord samples from ALS patients (around 40% loss, p<0.05). In contrast, chronic excitotoxicity in spinal cord increases DHA acid amount, with both enhanced concentrations of neuroprotective docosahexaenoic acid-derived resolvin D, and higher lipid peroxidation-derived molecules such as 8-iso-prostaglandin-F2-α (8-iso-PGF2α) levels. Since α-tocopherol improved mitochondrial respiratory function and motor neuron survival in these conditions, it is suggested that oxidative stress could boost motor neuron loss. Cell culture and metabolic flux experiments, showing enhanced expression of desaturases (FADS2) and β-oxidation enzymes after H2O2 challenge suggest that DHA production can be an initial response to oxidative stress, driven by TDP-43 aggregation and drebrin loss. Interestingly, these changes were dependent on cell type used, since human neurons exhibited losses of FADS2 and drebrin after oxidative stress. These features (drebrin loss and FADS2 alterations) were also produced by transfection by aggregation prone C-terminal fragments of TDP-43. CONCLUSIONS sALS is associated with tissue-specific DHA-dependent synthetic machinery alteration. Furthermore, excitotoxicity sinergizes with oxidative stress to increase DHA levels, which could act as a response over stress, involving the expression of DHA synthetic enzymes. Later on, this allostatic overload could exacerbate cell stress by contributing to TDP-43 aggregation. This, at its turn, could blunt this protective response, overall leading to DHA depletion and neuronal dysfunction.

Application of dried blood spot cards to determine olive oil phenols (hydroxytyrosol metabolites) in human blood

In this study, a fast and simple blood sampling and sample pre-treatment method based on the use of the dried blood spot (DBS) cards and ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) for the quantification of olive oil phenolic metabolites in human blood was developed and validated. After validation, the method was applied to determine hydroxytyrosol metabolites in human blood samples after the acute intake of an olive oil phenolic extract. Using the FTA DMPK-A DBS card under optimum conditions, with 20µL as the blood solution volume, 100µL of methanol/Milli-Q water (50/50, v/v) as the extraction solvent and 7 disks punched out from the card, the main hydroxytyrosol metabolites (hydroxytyrosol-3-O-sulphate and hydroxytyrosol acetate sulphate) were identified and quantified. The developed methodology allowed detecting and quantifying the generated metabolites at low μM levels. The proposed method is a significant improvement over existing methods to determine phenolic metabolites circulating in blood and plasma samples, thus making blood sampling possible with the volunteer pricking their own finger, and the subsequent storage of the blood in the DBS cards prior to chromatographic analysis.

Phytochemical Profiles of New Red-Fleshed Apple Varieties Compared with Traditional and New White-Fleshed Varieties

This study is an exhaustive chemical characterization of the phenolic compounds, triterpenes, and organic and ascorbic acids in red-fleshed apple varieties obtained by different breeding programs and using five traditional and new white-fleshed apple cultivars as reference. To carry out these analyses, solid-liquid extraction (SLE) and ultraperformance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) were used. The results showed that the red-fleshed apples contained, in either the flesh or peel, higher amounts of phenolic acids (chlorogenic acid), anthocyanins (cyanidin-3-O-galactoside), dihydrochalcones (phloretin xylosyl glucoside), and organic acids (malic acid) but a lower amount of flavan-3-ols than the white-fleshed apples. These quantitative differences could be related to an up-regulation of anthocyanins, dihydrochalcones, and malic acid and a down-regulation of flavan-3-ols (anthocyanin precursors) in both the flesh and peel of the red-fleshed apple varieties. The reported results should be considered preliminary because the complete phytochemical characterization of the red-fleshed apple cultivars will be extended to consecutive harvest seasons.

Seasonal Variability of the Phytochemical Composition of New Red-Fleshed Apple Varieties Compared with Traditional and New White-Fleshed Varieties

The main objective of this study was to evaluate the impact of the season on the apple phytochemical composition (phenolic compounds, triterpenes, and organic and ascorbic acids). For this proposal, four red-fleshed and five white-fleshed apple varieties from two consecutive seasons (2015 and 2016) were studied. A significant interaction with the season in some compounds was observed. The total phenolic content in the apple flesh from 2015 was higher than that from 2016 probably related with the lower rainfall during the harvest period in 2015 that could have favored hydric stress in the apple trees. The impact of the season on the apple skin was different. The 2016 season was characterized by higher maximum and minimum temperatures resulting in a higher content of flavonols, triterpenes, and organic acids. Anthocyanin concentration in both the flesh and skin of the red-fleshed apples showed no clear relationship to the season, and each variety showed an individual pattern.

Hydroxytyrosol: Emerging Trends in Potential Therapeutic Applications

Hydroxytyrosol (HT) and its derivatives represent the minor components of Virgin Olive Oil (VOO) that are of great interest for their pharmacological properties and among the most widely researched natural antioxidant compounds. In this review, the occurrence and metabolic fate of HT and its precursors are presented prior to discussing its beneficial effects on health. Bioavailability studies show that the metabolites detected in plasma depend on the model used (animal or human), the HT source (simple molecule or complex precursors) and the dose administered. However, in all cases HT sulphate appears to be the most ubiquitous metabolite in biofluids and it seems probable that it is responsible to a great extent for HT biological effects. Epidemiological evidence of HT and its derivatives against such lifestyle-associated pathologies as cancer, cardiovascular and neurodegenerative diseases is reviewed together with the newest perspectives on the mechanisms of action based on in-vitro and animal studies. According to the reviewed data, HT and its precursors could have the potential clinical use in cardiovascular diseases; more epidemiological data is needed to demonstrate their neurodegenerative diseases and cancer prevention.