Estrategias analíticas para la mejora de la determinación cualitativa y cuantitativa en metabolómica por espectrometría de masas

The basic objective of the research in this Thesis Book was to develop new analytical strategies based on the use of low- and high-resolution mass spectrometry to improve the detection and identification coverage in metabolomic analysis. These new strategies were applied throughout the main steps of the analytical process—sampling, sample preparation, determination and data analysis—and allowed improving basic analytical features such as sensitivity, selectivity and precision of metabolomic analysis methods (targeted and untargeted) and their detection capacity. The achievement of this basic objective has leaded to metabolomic analysis methods capable of providing a higher level of information, which is a key milestone for the resolution of biological problems. This objective was divided into three general objectives according to the different topics in this research: (i) to take benefit from the versatility of the triple quadrupole mass spectrometer to improve the identification/quantification of certain families of metabolites; (ii) to develop approaches to improve the detection and identification of metabolites by chromatographic techniques coupled to mass spectrometry in high resolution mode; and (iii) to create strategies for searching potential biomarkers in clinical and agro-food studies. From each general objective, we defined several concrete objectives: - To develop an automated qualitative and quantitative method based on on-line coupling of solid phase extraction (SPE) and liquid chromatography–tandem mass spectrometry (LC–MS/MS) to maximize sensitivity for determination of fatty acid esters of hydroxy fatty acids (FAHFAs) in serum (Chapter I). The method was further applied to a cohort of individuals to evaluate the influence of glycaemia on FAHFA levels. - To propose a qualitative/quantitative strategy for determination of polar lipids in human plasma by LC–MS/MS. Two MS/MS acquisition methods were combined to identify and confirm the presence of polar lipids in plasma (Chapter II). Thus, the process was carried out in two steps: (i) identification of lipids through the characteristic fragmentation pattern for each family; and b) confirmation of detected lipids by monitoring product ions corresponding to the fatty acids (FAs) conforming them or other characteristic product ions. - To study the differences at metabolite level between serum and plasma obtained with conventional tubes (heparin tube for plasma) and polymeric gel tubes by application of an untargeted approach based on gas chromatography coupled to time-of-flight mass spectrometry (GC–TOF/MS) (Chapter III). A cohort of volunteers was selected for blood sampling using four different tubes (plasma, plasma-gel, serum and serumgel). - To evaluate the influence of sample preparation on the determination of polar lipids in visceral adipose tissue (Chapter IV). Two different extractants were tested to compare their efficiency for the extraction of polar lipids, but also their inefficiency for extraction of acylglycerides (the main interference in the detection of polar lipids). Additionally, the implementation of an SPE step with a selective sorbent for retention of glycerophospholipids was assessed to check its influence on the subsequent detection of this family of lipids. - To maximize the identification coverage of metabolites found in pig fecal simples through the study of sample preparation (Chapter V). For this purpose, two analytical platforms such as LC–QTOF MS/MS and GC–TOF/MS were combined to evaluate their additivity in terms of identification. Concerning sample preparation, six solvents with different polarity were tested to evaluate the extraction performance and, in case of GC–MS, two derivatization protocols were compared. - To develop a new statistical package, called MetaboQC, to study and filtrate experimental variabililty in data sets generated by MS analysis of sequences processed for several days (Chapter VI). This new tool uses quality controls (QCs) to individually correct any tendency on quantitative signals of metabolites that can be associated to instrumental variability. - To study, by untargeted metabolomics analysis, the postprandial response to the oral fat tolerance test (OFTT) on plasma metabolomic profile (Chapter VII). Collected plasma samples were analyzed by LC–QTOF MS/MS and GC–TOF/MS. This test can open possibilities for the application of OFTT to the diagnostic of a wide range of pathologies. - To evaluate the predictive capacity of type 2 diabetes mellitus (T2DM) occurrence by examining the postprandial response (after OFTT) (Chapter VIII). With this aim, plasma samples were collected from 215 patients (CORDIOPREV project) at baseline and four hours after the OFTT. 107 individuals developed diabetes after five years. Collected plasma samples were analyzed by LC–QTOF MS/MS and GC–TOF/MS. - To elucidate the early events preceding the onset of islet autoimmunity and overt type 1 diabetes mellitus (T1DM). Metabolomics was used to determine levels of molecular lipids and polar metabolites in human peripheral blood mononuclear cells (PBMCs) isolated from prospective samples collected in the Type 1 Diabetes Prediction and Prevention (DIPP) study (Chapter IX). - To develop discrimination models and search for panels of markers with capability to classify slaughtered pigs by their feeding regime (Chapter X). 80 samples of subcutaneous adipose tissue from Iberian pigs subjected to four different feedings were used. Data were obtained from the classical method for the determination of FAs based on GC–FID and from a method for determination of carbon isotopic abundances by isotope ratio mass spectrometry (IRMS).El objetivo básico de la investigación en esta Tesis fue desarrollar nuevas estrategias analíticas basadas en el uso de espectrometría de masas de baja y alta resolución para mejorar la detección y la cobertura de identificación. Este objetivo se dividió en tres objetivos generales de acuerdo con los diferentes temas de esta investigación: (i) aprovechar la versatilidad del analizador de triple cuadrupolo (QqQ) para mejorar la identificación/cuantificación de ciertas familias de metabolitos; (b) desarrollar nuevas herramientas para mejorar la detección e identificación de metabolitos mediante técnicas cromatográficas acopladas a espectrometría de masas de alta resolución; y (c) crear estrategias para buscar biomarcadores potenciales en estudios clínicos y agroalimentarios. En este contexto, la Tesis ha dado lugar a los siguientes resultados: - Desarrollo de un método cualitativo y cuantitativo automatizado basado en el acoplamiento en línea de la extracción en fase sólida (SPE) y la cromatografía líquida con detección por espectrometría de masas en tándem (LC–MS/MS) para maximizar la sensibilidad en la determinación de ésteres de ácidos grasos y ácidos grasos hidroxilados (FAHFAs) en suero. El método se aplicó a una cohorte de individuos para evaluar la influencia de la glicemia en los niveles de FAHFAs [1]. - Propuesta de una estrategia cualitativa/cuantitativa para la determinación de lípidos polares en plasma humano por LC–MS/MS. Se combinaron dos métodos de adquisición de datos MS/MS para identificar y confirmar la presencia de lípidos polares en plasma. La propuesta se llevó a cabo en dos pasos: a) identificación de lípidos a través del patrón de fragmentación característico para cada familia; y b) confirmación de los lípidos detectados mediante la monitorización de iones producto correspondientes a los ácidos grasos (FAs) que los conforman u otros iones característicos. - Estudio sobre las diferencias a nivel de metabolitos entre suero y plasma obtenidos con tubos convencionales (tubo de heparina para plasma) y tubos con gel polimérico mediante la aplicación de un enfoque no dirigido basado en cromatografía de gases con detección por espectrometría de masas con analizador de tiempo de vuelo (GC– TOF/MS). Se seleccionó una cohorte de voluntarios para el muestreo de sangre utilizando cuatro tipos de tubos (plasma, plasma-gel, suero y suero-gel) [2]. - Evaluación de la influencia de la preparación de la muestra en la determinación de lípidos polares en tejido adiposo visceral. Se probaron dos disolventes para comparar su eficiencia en la extracción de lípidos polares, pero también su ineficiencia para la extracción de acilglicéridos (las principales interferencias en la detección de lípidos polares). Además, se evaluó la implementación de una etapa SPE con un sorbente selectivo para la retención de glicerofosfolípidos con el fin de verificar su influencia en la detección posterior de esta familia de lípidos [3]. - Desarrollo de una estrategia para maximizar la cobertura de metabolitos identificados en muestras fecales de cerdo a través del estudio de la preparación de la muestra. Con este propósito se combinaron dos técnicas de detección, LC–QTOF MS/MS y GC–TOF/MS, para evaluar su complementariedad en términos de identificación. Con respecto a la preparación de la muestra, se probaron seis disolventes con diferente polaridad para evaluar su rendimiento de extracción y, en el caso de GC–MS, se compararon dos protocolos de derivatización [4]. Diseño y desarrollo de un nuevo paquete estadístico, llamado MetaboQC, para estudiar y filtrar la variabilidad instrumental en conjuntos de datos generados mediante análisis por espectrometría de masas en secuencias desarrolladas durante varios días. Esta nueva herramienta utiliza controles de calidad (QCs) para corregir individualmente cualquier tendencia en las señales cuantitativas de metabolitos que puedan estar asociadas a la variabilidad instrumental [5]. - Estudio, mediante un análisis metabolómico no dirigido, de la respuesta posprandial a la prueba oral de tolerancia a la grasa (OFTT) en el perfil metabólico plasmático. Las muestras de plasma recolectadas se analizaron por LC–QTOF MS/MS y GC– TOF/MS. Los resultados de este estudio abren una vía al uso de este test para el diagnóstico de un amplio abanico de patologías. - Evaluación de la capacidad predictiva de la aparición de diabetes mellitus tipo 2 (T2DM) mediante el examen de la respuesta posprandial (después de la OFTT). En este estudio se recogieron muestras de plasma de 215 pacientes (proyecto CORDIOPREV) justo antes y cuatro horas después de la prueba OFTT. 107 personas desarrollaron diabetes después de cinco años. Las muestras de plasma se analizaron por LC–QTOF MS/MS y GC–TOF/MS. - Dilucidación de los eventos que preceden al inicio de la autoinmunidad de los islotes y la diabetes mellitus tipo 1 (T1DM). Se utilizó la metabolómica para determinar los niveles de lípidos moleculares y metabolitos polares en células mononucleares de sangre periférica humana (PBMC) aisladas de muestras prospectivas recolectadas en el estudio de Predicción y Prevención de Diabetes Tipo 1 (DIPP) [6]. - Desarrollo de modelos de discriminación y búsqueda de paneles de marcadores con capacidad para la clasificación de cerdos por su régimen de alimentación. Se utilizaron 80 muestras de tejido adiposo subcutáneo de cerdos ibéricos sometidos a cuatro regímenes de alimentación. Los datos se obtuvieron combinando el método clásico para la determinación de FAs basado en GC–FID y un método para la determinación de las abundancias isotópicas de carbono por espectrometría de masas con relación isotópica (IRMS) [7]

Promotion of self-employed work in the laboratory practices of students of the degree in chemistry: case study

En este estudio se ha perseguido incrementar la participación de los alumnos en las prácticas de laboratorio. El motivo principal ha sido evitar que el alumno memorice los conceptos teóricos sin ser capaz de aplicarlos a la resolución de problemas reales, y así adquiera compentencias útiles para cuando se introduzca en el mundo laboral. La nueva metodología se desarrolló en una asignatura optativa del Grado de Química. Para abordar la nueva metodología propuesta en este proyecto, los alumnos elaboraron los protocolos de tres prácticas, sobre tres temas propuestos por el profesorado. Una vez supervisados dichos protocolos por el profesorado, los alumnos las realizaron de forma autónoma en el laboratorio. La evaluación de los alumnos se realizó mediante: elaboración de los protocolos de prácticas, realización de las prácticas de laboratorio (mediante rúbrica) y cuestionario sobre conceptos teóricos (mediante la herramienta Kahoot). Finalmente, se evaluó el grado de aceptación de la nueva metodología docente por parte del alumnado, para detectar aspectos a mejorar para el próximo año académico. Esta metodología ha permitido una mayor implicación del alumnado en las sesiones de laboratorio, ya que pudieron aplicar los conceptos teóricos adquiridos sobre Química Analítica y adquirir competencias, tales como capacidad de aprendizaje autónomo.In this study we have sought to increase the participation of students in laboratory practices. The main reason has been to prevent the student from memorizing the theoretical concepts without being able to apply them to the resolution of real problems, and thus acquire useful competences for when entering into the working world. The new methodology was developed in an optional subject of the Degree in Chemistry. Therefore, it was proposed that they themselves elaborate the protocols of practices, on subjects proposed by the teaching staff, and that they carried them out independently in the laboratory. The evaluation of the students' work was carried out through three activities: preparation of the practical protocols, carrying out of the laboratory practices (using a rubric) and questionnaire on theoretical concepts (using the Kahoot tool). Finally, the degree of acceptance of the new teaching methodology by the students was evaluated. This allowed to detect the aspects to improve for the next academic year. This methodology has allowed a greater involvement of the students in the laboratory sessions, since they could apply the theoretical concepts acquired on Analytical Chemistry and acquire competences, such as autonomous learning capacity

Early Salmonella Typhimurium infection in pigs disrupts Microbiome composition and functionality principally at the ileum mucosa

[EN] Salmonella is a major foodborne pathogen which successfully infects animal species for human consumption such as swine. The pathogen has a battery of virulence factors which it uses to colonise and persist within the host. The host microbiota may play a role in resistance to, and may also be indirectly responsible from some of the consequences of, Salmonella infection. To investigate this, we used 16S rRNA metagenomic sequencing to determine the changes in the gut microbiota of pigs in response to infection by Salmonella Typhimurium at three locations: ileum mucosa, ileum content and faeces. Early infection (2 days post-infection) impacted on the microbiome diversity at the mucosa, reflected in a decrease in representatives of the generally regarded as desirable genera (i.e., Bifidobacterium and Lactobacillus). Severe damage in the epithelium of the ileum mucosa correlated with an increase in synergistic (with respect to Salmonella infection; Akkermansia) or opportunistically pathogenic bacteria (Citrobacter) and a depletion in anaerobic bacteria (Clostridium spp., Ruminococcus, or Dialliser). Predictive functional analysis, together with metabolomic analysis revealed changes in glucose and lipid metabolism in infected pigs. The observed changes in commensal healthy microbiota, including the growth of synergistic or potentially pathogenic bacteria and depletion of beneficial or competing bacteria, could contribute to the pathogen's ability to colonize the gut successfully. The findings from this study could be used to form the basis for further research aimed at creating intervention strategies to mitigate the effects of Salmonella infectionSIWe want to acknowledge the staff from the Genomics and Animal breeding group at the University of Córdoba for their technical support to carry out this study. This article is based upon work from COST Action FA1401 (PiGutNet), supported by COST (European Cooperation in Science and Technology. This work was supported by the Spanish Ministry of Economy and Competitiveness (AGL2014-54089-R/AGL2017-87415-R). HA was funded by the PiGutNet COST action (FA1401) for a Short-Term Scientific Mission at INRA’s GABI laboratory (Jouy-en- Josas, France). SZL is a postdoctoral researcher supported by the Postdoctoral Trainee Program of the Spanish Ministry of Economy and Competitiveness (FPDI-2013-15619). HA is a postdoctoral researcher supported by the Juan de la Cierva Postdoctoral Trainee Program of the Spanish Ministry of Economy and Competitiveness (FJCI-2014-22877

Metabolic alterations in immune cells associate with progression to type 1 diabetes

Aims/hypothesisPrevious metabolomics studies suggest that type 1 diabetes is preceded by specific metabolic disturbances. The aim of this study was to investigate whether distinct metabolic patterns occur in peripheral blood mononuclear cells (PBMCs) of children who later develop pancreatic beta cell autoimmunity or overt type 1 diabetes.MethodsIn a longitudinal cohort setting, PBMC metabolomic analysis was applied in children who (1) progressed to type 1 diabetes (PT1D, n = 34), (2) seroconverted to ≥1 islet autoantibody without progressing to type 1 diabetes (P1Ab, n = 27) or (3) remained autoantibody negative during follow-up (CTRL, n = 10).ResultsDuring the first year of life, levels of most lipids and polar metabolites were lower in the PT1D and P1Ab groups compared with the CTRL group. Pathway over-representation analysis suggested alanine, aspartate, glutamate, glycerophospholipid and sphingolipid metabolism were over-represented in PT1D. Genome-scale metabolic models of PBMCs during type 1 diabetes progression were developed by using publicly available transcriptomics data and constrained with metabolomics data from our study. Metabolic modelling confirmed altered ceramide pathways, known to play an important role in immune regulation, as specifically associated with type 1 diabetes progression.Conclusions/interpretationOur data suggest that systemic dysregulation of lipid metabolism, as observed in plasma, may impact the metabolism and function of immune cells during progression to overt type 1 diabetes.</p

Improving <i>Tenebrio molitor</i> Growth and Nutritional Value through Vegetable Waste Supplementation

Huge amounts of vegetable wastes are generated by the food industry. Their bioconversion into valuable products (e.g., insect flours or biofertilizer) through insect farming is a promising solution to reduce their negative environmental and economic impacts. This study evaluates the growth of Tenebrio molitor larvae and their nutritional profile after supplementing their diets with vegetable wastes. Over a 6-week period, 45-day larvae were fed a diet comprising wheat bran supplemented (1:1) with cucumber or tomato wastes from both conventional and ecological crops. The control diet consisted of wheat bran and an equivalent amount of water to compensate for the waste moisture. Larval weight was measured weekly, and length measures were taken fortnightly. Nutritional composition and fatty acid profile were analyzed at the end of the study in 90-day larvae. Regardless of using vegetable waste from conventional or ecological harvesting, the weight of 6-week supplemented larvae almost doubled that of larvae fed with just wheat bran, and their length was 15% higher. Supplementation also increased larval polyunsaturated fatty acid percentage by 22–37%, with linoleic acid being the most abundant. Likewise, larval protein content reached 50% after supplementation. This study demonstrates that both cucumber and tomato wastes from conventional or ecological crops are excellent supplements for T. molitor’s diet, improving their nutritional value and reducing the time necessary for larvae growth

Fomento del trabajo autónomo en las prácticas de laboratorio de alumnos del grado de química: caso de estudio

En este estudio se ha perseguido incrementar la participación de los alumnos en las prácticas de laboratorio. El motivo principal ha sido evitar que el alumno memorice los conceptos teóricos sin ser capaz de aplicarlos a la resolución de problemas reales, y así adquiera compentencias útiles para cuando se introduzca en el mundo laboral. La nueva metodología se desarrolló en una asignatura optativa del Grado de Química. Para abordar la nueva metodología propuesta en este proyecto, los alumnos elaboraron los protocolos de tres prácticas, sobre tres temas propuestos por el profesorado. Una vez supervisados dichos protocolos por el profesorado, los alumnos las realizaron de forma autónoma en el laboratorio. La evaluación de los alumnos se realizó mediante: elaboración de los protocolos de prácticas, realización de las prácticas de laboratorio (mediante rúbrica) y cuestionario sobre conceptos teóricos (mediante la herramienta Kahoot). Finalmente, se evaluó el grado de aceptación de la nueva metodología docente por parte del alumnado, para detectar aspectos a mejorar para el próximo año académico. Esta metodología ha permitido una mayor implicación del alumnado en las sesiones de laboratorio, ya que pudieron aplicar los conceptos teóricos adquiridos sobre Química Analítica y adquirir competencias, tales como capacidad de aprendizaje autónomo