Analysis of urinary exosomal metabolites identifies cardiovascular risk signatures with added value to urine analysis

Background: Subclinical atherosclerosis may result in fatal cardiovascular (CV) events, but the underlying mechanisms and molecular players leading to disease are not entirely understood. Thus, novel approaches capable of identifying the factors involved in pathological progression and providing a better understanding of the subjacent mechanisms are needed. Extracellular vesicles (EVs) have been shown to have numerous biological functions, and their metabolome has recently generated interest as a source of novel biomarkers. The metabolic content of the exosomes has been so far unexplored in cardiovascular disease (CVD), and here, we developed an analytical strategy aimed at probing urinary exosomal metabolite content and its association to CV risk. Results: Direct analysis of the exosomes without metabolite extraction was evaluated by high-resolution magic angle spinning (1 H HR-MAS). Other two methodologies for the analysis of exosomal metabolites by 1 H NMR were set up, based on methanol or organic solvents sequential extraction. The three methods were compared in terms of the number of detected signals and signal to noise ratio (S/N). The methanol method was applied to identify altered metabolites in the urinary exosomes of subjects with programmed coronary artery by-pass grafting (CABG) versus a control group. Target mass spectrometry (MS) was also performed for differential analysis. The clinical performance of exosomal metabolites of interest in CVD was investigated, and the added value of the exosomes compared to urine analysis was evaluated. Based on S/N ratio, simplicity, reproducibility, and quality of the spectrum, the methanol method was chosen for the study in CVD. A cardiometabolic signature composed by 4-aminohippuric acid, N-1-methylnicotinamide, and citric acid was identified in urinary exosomes. Directly in urine, 4-aminohippuric acid and citric acid do not show variation between groups and changes in N-1-methylnicotinamide are less pronounced, proving the added value of exosomes. Conclusions: We set up a novel methodology to analyze metabolic alterations in urinary exosomes and identified a cardiometabolic signature in these microvesicles. This study constitutes the first evidence of a role for the exosomal metabolism in CVD and demonstrates the possibility to evaluate the urinary exosomal metabolic content by NMR and MS

Nuevas aplicaciones sintéticas de los cationes trifliloxicarbenio

Esta memoria se enmarca dentro de una línea de investigación en la que viene abordando la reactividad y aplicaciones sinteticas de los compuestos carbonilicos frente al anhidrodo trifluormetanosulfonico, (tf2o). De acuerdo con ello, se plantearon los siguientes objetivos: A) estudiar la reactividad de compuestos carbonilicos funcionalizados con tf2o, así como la naturaleza de los intermedios implicados. B) aplicación de las potenciales utilizadas sintéticas de dichos intermedios para la síntesis de heteociclos funcionalizados. El estudio realizado nos ha permitido obtener resultados muy diferentes dependiendo de la naturaleza del sustrato de partida y de las condiciones experimentales. Por un lado, se ha comprobado la existencia de un carbocation trifliloxicarbenio como intermedio del proceso, originado por reacción del tf2o con el o carbonilico, de forma análoga a lo que sucede en el caso de sustratos no funcionalizados. Por otro lado, se han desarrollado nuevas vías sintéticas para la preparación de 5-halopirimidinas, 4-alcoxipirimidinas, 3-alcoxiisoquinolinas, gamma-pironas. También se ha puesto a punto un procedimiento de sintesis de diarilcetonas por acilacion a partir de esteres, a través de la formación de triflatos de acilo

Poloxamine/D-α-Tocopheryl polyethylene glycol succinate (TPGS) mixed micelles and gels:Morphology, loading capacity and skin drug permeability

The combination of polymeric surfactants with different features into mixed micelles give access to properties that may be superior to the single-component micelles. In this work, we investigated synergistic effects in mixtures of D-α-Tocopheryl polyethylene glycol succinate (TPGS) with poloxamines (also known as Tetronic), pH-responsive and thermogelling polyethylene oxide (PEO)-polypropylene oxide (PPO) 4-arm block copolymers. We examined the morphology of the self-assembled micelles of TPGS with Tetronic 1107 (T1107) and 908 (T908) in the presence of naproxen (NA), used as a model drug, and assessed the capacity of the single and mixed micelles to trap the guest, using a combination of small-angle neutron scattering (SANS) and NMR spectroscopy (1D, 2D-NOESY and diffusion NMR), over a range of compositions and temperatures, in the dilute regime and gel state. NA did not interact with T1107 or T908 in their unimer form, but it was incorporated into the hydrophobic core of the micelles above the critical micellar temperature (CMT). In contrast, TPGS dissolved NA at any temperature, mainly in the tocopherol core, with some partitioning in the PEG-shell. The micellar structure was not altered by the presence of NA, except for an expansion of the core size, a result of the preferential accumulation of NA in that compartment. The solubility of the drug in single component micelles increased markedly with temperature, while mixed micelles produced an intermediate enhancement of the solubility between that of TPGS and the poloxamines, which increased at higher TPGS/poloxamine ratios. Micellar hydrogels formed by the packing of the polymeric mixed micelles in a BCC macrolattice, whose structure was not altered by the presence of the drug (at least at 0.2 wt%). The applicability of the drug-loaded gels for topical formulations was explored by transdermal diffusion testing using a synthetic model of skin, showing that the diffusion of NA across the membrane was enhanced by incorporating small amounts of TPGS to the hydrogel, especially with the more hydrophilic T908

A 1H NMR-based protocol for the detection of refined olive oil adulterations with refined hazelnut oil

A 1H NMR analytical protocol for the detection of refined hazelnut oils in admixtures with refined olive oils is reported according to ISO format. The main purpose of this research activity is to suggest a novel analytical methodology easily usable by operators with a basic knowledge of the NMR spectroscopy. The protocol, developed on 92 oil samples of different origin within the European MEDEO project (Development and Assessment of Methods for the Detection of Adulteration of Olive Oil with Hazelnut Oil) (1,2), is based on 1H NMR measurements combined with a suitable statistical analysis. It was developed using a 600 MHz instrument and was tested by two independent laboratories on 600 MHz spectrometers allowing the detection down to 10% adulteration of olive oil with refined hazelnut oils. Finally, the potentiality and the limitations of the protocol applied on spectrometers operating at different magnetic fields i.e. at the proton frequency of 500 MHz and 400 MHz were investigated.JRC.DDG.I.6-Systems toxicolog