1. Investigación

Short-chain fatty acids (SCFAs) are organic saturated monocarboxylic acids with fewer than six carbon atoms. Their recently described relevance in health and disease has brought to the table the need for a convenient method for their analysis. The current research presents an optimized and validated LC-QqQ-MS method for the absolute quantification of SCFAs in plasma and feces. This method has proven to be accurate and precise in a wide range of concentrations (0.2 – 200 µM). It has been applied to more than 500 samples showing good precision, reproducibility, and linearity results. The method was applied to study samples from anorexia nervosa (AN) patients and matched healthy controls (HC). The results showed significant differences in the concentration of plasmatic and fecal SCFAs between both groups whose implications in AN are yet to be determined. Nonetheless, these findings denote important alterations in the SCFAs homeostasis in AN which can be linked to the metabolic alterations and the intestinal dysbiosis already characterized in the disease. More research is still required to identify the function of these metabolites in the pathophysiology of AN, but the present work provides a reliable methodology and valuable information to continue delving into the potential role of the microbiota as a therapeutic target via the gut-brain axis.

Birth presents a metabolic challenge to cardiomyocytes as they reshape fuel preference from glucose to fatty acids for postnatal energy production1,2. This adaptation is triggered in part by post-partum environmental changes3, but the molecules orchestrating cardiomyocyte maturation remain unknown. Here we show that this transition is coordinated by maternally supplied γ-linolenic acid (GLA), an 18:3 omega-6 fatty acid enriched in the maternal milk. GLA binds and activates retinoid X receptors4 (RXRs), ligand-regulated transcription factors that are expressed in cardiomyocytes from embryonic stages. Multifaceted genome-wide analysis revealed that the lack of RXR in embryonic cardiomyocytes caused an aberrant chromatin landscape that prevented the induction of an RXR-dependent gene expression signature controlling mitochondrial fatty acid homeostasis. The ensuing defective metabolic transition featured blunted mitochondrial lipid-derived energy production and enhanced glucose consumption, leading to perinatal cardiac dysfunction and death. Finally, GLA supplementation induced RXR-dependent expression of the mitochondrial fatty acid homeostasis signature in cardiomyocytes, both in vitro and in vivo. Thus, our study identifies the GLA–RXR axis as a key transcriptional regulatory mechanism underlying the maternal control of perinatal cardiac metabolism.

The characterization of specialized cell subpopulations in a heterogeneous tissue is essential for understanding organ function in health and disease. A popular method of cell isolation is fluorescence-activated cell sorting (FACS) based on probes that bind surface or intracellular markers. In this study, we analyze the impact of FACS on the cell metabolome of mouse peritoneal macrophages. Compared with directly pelleted macrophages, FACS-treated cells had an altered content of metabolites related to the plasma membrane, activating a mechanosensory signaling cascade causing inflammation-like stress. The procedure also triggered alterations related to energy consumption and cell damage. The observed changes mostly derive from the physical impact on cells during their passage through the instrument. These findings provide evidence of FACS-induced biochemical changes, which should be taken into account in the design of robust metabolic assays of cells separated by flow cytometry.

Increased caloric intake associated with decreased physical activity and the presence of thrifty genes that are theoretically adapted to enhance the energy storage efficiency, cause metabolic changes that result in diet-related diseases or disorders. Such phenotypes are prevalent in populations of developed countries and their incidence is continuing to rise. Therefore, early diagnosis of diet-related diseases is an exciting field of research. The application of ‘omics’ technology, particularly metabolomics, has revealed the metabolic changes associated to diet-related diseases and also consequences of diet intervention in a global un-targeted way. The on-going development of dietary ideal models could elucidate the sequence of events, starting with the interaction between dietary habits and genetic adaptations that cause the metabolic changes induced as well as auxiliary symptoms and associated diseases. In this review, a range of mass spectrometry techniques applied to metabolomics of diet related diseases is discussed, including the combination of metabolomics with other studies to reveal systems properties of the diseases. Since it is difficult to set up a clinical study based on the probability of finding exploratory biomarkers to be applied in wide-population screening, many metabolomics studies have revealed biomarkers of 31 the complications of the disease, which could have power as prognostic biomarkers.

Background: Cardiac steatosis and apoptosis are key processes in diabetic cardiomyopathy, but the underlying mechanisms have not been elucidated, leading to a lack of effective therapy. The mineralocorticoid receptor blocker, eplerenone, has demonstrated anti-fibrotic actions in the diabetic heart. However, its effects on the fatty-acid accumulation and apoptotic responses have not been revealed. Methods: Non-hypertensive Zucker Diabetic Fatty (ZDF) rats received eplerenone (25 mg/kg) or vehicle. Zucker Lean (ZL) rats were used as control (n = 10, each group). After 16 weeks, cardiac structure and function was examined, and plasma and hearts were isolated for biochemical and histological approaches. Cultured cardiomyocytes were used for in vitro assays to determine the direct effects of eplerenone on high fatty acid and high glucose exposed cells. Results: In contrast to ZL, ZDF rats exhibited hyperglycemia, hyperlipidemia, insulin-resistance, cardiac steatosis and diastolic dysfunction. The ZDF myocardium also showed increased mitochondrial oxidation and apoptosis. Importantly, eplerenone mitigated these events without altering hyperglycemia. In cultured cardiomyocytes, high-concentrations of palmitate stimulated the fatty-acid uptake (in detriment of glucose assimilation), accumulation of lipid metabolites, mitochondrial dysfunction, and apoptosis. Interestingly, fatty-acid uptake, ceramides formation and apoptosis were also significantly ameliorated by eplerenone. Conclusions: By blocking mineralocorticoid receptors, eplerenone may attenuate cardiac steatosis and apoptosis, and subsequent remodelling and diastolic dysfunction in obese/type-II diabetic rats.

Pulmonary embolism (PE) is a common cardiovascular emergency which can lead to pulmonary hypertension (PH) and right ventricular failure as a consequence of pulmonary arterial bed occlusion. The diagnosis of PE is challenging due to non-specific clinical presentation what results in relatively high mortality. Moreover, the pathological factors associated with PE are poorly understood. Metabolomics can provide with new highlights which can help in the understanding of the processes and even propose biomarkers for its diagnosis. In order to obtain more information about PE and PH, acute PE was induced in large white pigs and plasma was obtained before and after induction of PE. Metabolic fingerprints from plasma were obtained with LC-QTOF-MS (positive and negative ionization) and GC-Q-MS. Data pretreatment and statistical analysis (uni- and multivariate) was performed in order to compare metabolic fingerprints and to select the metabolites that showed higher loading for the classification (28 from LC and 19 from GC). The metabolites found differentially distributed among groups are mainly related to energy imbalance in hypoxic conditions, such as glycolysis-derived metabolites, ketone bodies, and TCA cycle intermediates, as well as a group of lipidic mediators that could be involved in the transduction of the signals to the cells such as sphingolipids and lysophospholipids, among others. Results presented in this report reveal that combination of LC−MS- and GC−MS-based metabolomics could be a powerful tool for diagnosis and understanding pathophysiological processes due to acute PE.

The metabolome is the complete set of small molecules coming from protein activity (anabolism and catabolism) in living systems. They have a broad range of chemical structures and physico-chemical properties and therefore different analytical methodologies are necessary. Highly polar metabolites, such as sugars and most amino acids are not retained by conventional reversed phase liquid (RP-LC) chromatography columns. Without sufficient retention, coelution may result in identification problems while the detection of compounds by mass spectrometry at low concentrations may also be problematic due to ion suppression. In order to retain compounds based on their hydrophilicity, polar stationary phases and hydrophilic interaction liquid chromatography (HILIC) provide a complementary tool to RP-LC for untargeted comprehensive metabolite fingerprinting. However, robustness of the methods is still limiting their applications. This review focuses on sample pre-treatment, stationary phases, analytical methods and applications for polar compound analysis in biological matrices.

Se ha administrado una dieta semisintética conteniendo un 10% de aceite de oliva o de pescado como único componente graso no-vitamínico a ratas preñadas, que fueron estudiadas al día 20 de gestación. En plasma de las madres, los porcentajes de ácidos grasos saturados fueron similares en ambos grupos, el de ácido oleico y de ácido araquidónico fueron superiores en las de aceite de oliva, mientras que los de ácidos eicosapentaenoico y docosahexaenoico fueron superiores en las de aceite de pescado. En el plasma de los fetos, los cambios fueron similares a los de las madres, mostrando una insuficiencia de ácido araquidónico en los de madres alimentadas con aceite de pescado. Tanto en plasma como en hígado de los fetos, la concentración de vitamina E fué inferior en los de madres alimentadas con dieta de aceite de pescado que con la de aceite de oliva. A su vez, la respuesta lipolítica de los adipocitos a un agonista b3-adrenérgico fue inferior en ratas preñadas que en vírgenes, pero el efecto inhibidor que produce en éstas el anclaje euglucémico e hiperinsulinémico desaparece cuando son alimentadas con dieta de aceite de pescado. Estos resultados muestran que un exceso en ácidos grasos poliinsaturados en la dieta durante la gestación en la rata, como el producido cuando ésta contiene aceite de pescado en vez de aceite de oliva, da lugar a una deficiencia en ácido araquidónico y vitamina E en los fetos y altera la normal respuesta lipolítica del tejido adiposo de la madre, teniendo consecuencias que podrían ser indeseables para el desarrollo postnatal.

New polar reversed-phase stationary phases in HPLC provide specific selectivities which can help to solve traditional chromatographic problems related to the development of chromatographic methods with widely different retention times for the sample components. One such case is the analysis of pharmaceutical formulations against the common cold. Acetaminophen, phenylephrine and chlorpheniramine, compounds with different polarities, are frequently associated in these drugs. An isocratic and rapid HPLC method for the simultaneous determination of the three compounds, acetaminophen, phenylephrine and chlorpheniramine, in capsules as pharmaceutical formulations, including the separation of impurities (4-aminophenol and 4-chloracetanilide) and excipients, has been developed and validated. The final chromatographic conditions employed a Supelco Discovery HS PEG column poly(ethyleneglycol) 1530.46 cm, 5 mm. The mobile phase was 20 mM phosphate buffer, pH 7.0–acetonitrile (90:10, v/v) at a flow-rate of 1 ml/min. UV detection was performed at 215 nm for all the compounds except acetaminophen, which was measured at 310 nm. Validation parameters permit us to consider this method suitable.

An HPLC method for Vitamins A and E in rat plasma has been developed. The main goals of the method are the small amount of sample, 50l, and the direct extraction of analytes in one step with acetone, which is a solvent compatible with the reverse-phase mobile phases. Recoveries, as compared with classical and more tedious methods, were near 100%. The method employs a Supelco Discovery® C18 column and methanol/water (95:5, v/v) as mobile phase. After being developed, the method was validated following ICH guidelines, with UV, fluorescence and electrochemical detectors. It proved to be selective, lineal, accurate and precise. This method greatly simplifies sample treatment and that is a critical point when working with a large number of samples.