“As part of the CardioSCOPE project, it was necessary to travel to Biosphere Teslab (Reus, Spain), where we analyzed murine plasma samples. Samples were obtained in our Gene Regulation and Molecular Therapy laboratory (ICBP NS) from control atherosclerotic mice and mice injected with adenovirus expressing ApoA2. During this secondment, we performed laboratory analyses, in which the concentration of lipids was determined in plasma samples from two types of mice: apoE-deficient and LDLR-deficient. The concentration of triglycerides and cholesterol in the plasma of mice was determined by turbidimetry analysis. Turbidimetry is the technique of evaluating the decline in transmitted light intensity caused by the scattering effect of particles suspended in the medium. For this analysis, I went to the Lipids and Arteriosclerosis Research Unit (URLA), a collaborating part of Biospher Teslab. Also, within URLA, I participated in the preparation of human serum samples for ApoC3 testing by ELISA, a project carried out by the Biosfer Teslab company.
Also, by NMR spectroscopy we analyzed the ratio between lipoproteins, the amount of low molecular weight metabolites, glycoproteins and other lipids both in total plasma and only in the lipid fraction. Lipids were previously extracted following a protocol based on the Matyash method.
Matyash extraction simplifies the extraction process by collecting lipids in the upper phase. I participated in the development of a series of lipid extractions from human feces, as part of the medical services offered by this company, in order to obtain clinical results.
Together with the Biosphere Teslab team, we examined the results obtained from murine plasma samples, obtained by NMR spectroscopy. I had the opportunity to see and understand bio-informatic analysis and analyzed the obtained spectra. I started to discover the program R studio, a data analysis program in a programming code, something completely new to me.
I also had the opportunity to visit the Center of Omics Science in Tarragona, where I observed both the equipment and a segment of the data processing procedures. I understood the principle of high capacity NMR spectroscopy, the importance and applicability of metabolomics. The basic principle behind NMR spectroscopy is that nuclei exhibit different nuclear spin states when subjected to an external magnetic field. NMR detects transitions between these spin states, which are unique to the specific nuclei under consideration and influenced by the environment of those nuclei.
This secondment enriched my experience with more theoretical and practical knowledge regarding work in the laboratory and experimental organization.”
Florina-Irina Tudorache, PhD student from ICBP “Nicolae Simionescu”, Bucharest
“During the secondment, we discussed with Dr. Núria Amigo about the project the lab is involved in and we performed some experiments by using metabolomic techniques.
First, we assessed the concentration of total cholesterol and triglycerides by turbidimetry. Second, we performed NMR spectrometry for the lipidic profile and low molecular weight metabolites (LMWM) panel.
Turbidimetry is a method based on the scattering or absorption of light by solid or colloidal particles suspended in solution. The measurement of the intensity of transmitted light is a function of the concentration of dispersed phase. The intensity of transmitted light decreases due to presence of non transparent particles in the sample.
NMR spectrometry is a very complex method that can provide information on the structure of the molecule and also determine the content and purity of one sample. The priciple of this method is based on the ability of nuclei of a molecule to acquire a higher energy level when an external magnetic field is supplied. Many nuclei have spin and all nuclei are electrically charged. Proton (1H) NMR is one of the most widely used NMR methods by organic chemists. The protons present in the molecule will behave differently depending on the surrounding chemical environment, making it possible to elucidate their structure. For obtaining the lipid profile of our samples by NMR spectrometry, the lipids were extracted using the modified method of Matyash and collaborators (2008). The assay was performed using an Agilent Bravo Automated machine. The method uses a non polar solvent- diisopropyl ether (DIIPE) and two semipolar solvents -buthanol and ethyl acetate. Phase separation is induced by adding distilated water. Lipids are aspirated from the upper phase, dried and used for the NMR spectrometry (Bruker Ultrashield 600 PLUS machine) when needed.
After obtaining the raw data from all the experiments perfomed, I started processing it and analysing it statistically using R. I created density and violin plots for each metabolite analysed and also an ANOVA test was run.
During a visit at the Center of Omics in Tarragona, I saw the mass spectrometry equipment and I got a better understanding of the main differences between the types of mass spectrometry.
Personally, I find this experience beautiful, intriguing and helpful as I was introduced in the fascinating world of biostatistics. As expected, this secondment gave me the opportunity to embetter my knowledge regarding metabolomics (including experiments and data analysis) and also to meet and work with a very open and professional team we could establish a fruitful collaboration with.”
Gabriela Ciumeica, PhD student from ICBP “Nicolae Simionescu”, Bucharest