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Assessment of Embryo Culture Media Metabolome by Raman and Infrared Spectroscopy

Metabolomics is a scientific discipline that studies chemical processes involving intermediates and products of metabolism (metabolites). The field of metabolomics most commonly includes the study of global metabolite profiles in a system (cell, tissue or organism), the systematic study of the unique chemical fingerprints that specific cellular processes leave behind, specifically, the study of their small-molecule metabolite profiles and the systematic analysis of the inventory of metabolites (small-molecule biomarkers) that represent the functional phenotype at the cellular level. The metabolome refers to the complete inventory of small-molecule, non-proteinaceous compounds, such as metabolic intermediates, ATP, fatty acids, glucose, cholesterol, hormones and other signaling molecules, as well as secondary metabolites that are found within a biological sample. As such, the metabolome changes continuously, depending upon the activation and interaction of the various metabolic pathways within the cell. In addition, the metabolome is considered to be a reflection of phenotype, which can then be used to infer gene function. Metabolomics arise from genomics, transcriptomics and proteomics, and due to its simplicity and inexpensiveness (relative to aforementioned disciplines) became popular method for research in biology and biomedicine. While genomics and proteomics can provide important information on the expected function, metabolomics provides an immediate snapshot of all current biological functions reflecting up-to-the-minute events. By systematically measuring the population of small-molecule biomarkers (metabolites), scientists can establish profiles or signatures of healthy individuals versus those with specific illnesses. Moreover, metabolomics can provide indications of a metabolic problem or lesion with high accuracy and at lower costs than genomics, transcriptomics or proteomics, and is therefore well suited for widespread investigations in the life sciences. As such, metabolomics is expected to be a relevant tool in the management of various medical conditions and, therefore, an important method for studying basic biological functions in conjunction with other methods. Studying complex metabolic profiles of biological systems requires specific analytical techniques. There are several different means of detection that can be used to obtain metabolomic data. While mass spectrometry and nuclear magnetic resonance are by far the two leading technologies used for metabolomics research, Raman and infrared spectroscopy have also been successfully employed to identify and quantify biomarkers. These latter methods have similar levels of analytical sensitivity to the previous methods, but also have several added advantages such as the direct sample measurement in which no preparation is required, the little chemical bias, smaller instrument size and easier maintenance of instruments as well as the rapid, simultaneous analysis of multiple biomarkers. In this work we analyzed culture media in which rat embryos grew for 14 days, with and without valproate (VPA) which is a chemical compound used in medicine for the prevention of migraine headaches and in the treatment of epilepsy and bipolar disorder. Using FTIR spectroscopy the spectra of media metabolome were recorded in order to determine whether this method could be suitable for assessing teratogenic effect of different substances on embryo growth. The recorded spectra were analyzed by statistical methods of PC analysis and PC regression. It was shown that the method of FTIR spectroscopy can distinguish media metabolome between control group in which rat embryos grew without valproate and the one in which they grew with valproate which proved that valproat toxicity is reflected in the change of the metabolome of the media, which can be monitored by FTIR spectra. We have established that the spectroscopic analysis of the metabolome media contains information about the difference in the growth and development of embryos through the monitored period of time. Using PCA analysis we have shown that from metabolome spectra we can determine for how long the embryos grew in culture. These results suggest that FTIR spectroscopic analysis of the media metabolome can be a new model for the screening of different embryo toxic and teratogenic substances. We are now in a process of repeating the same experiment using Raman spectroscopy as a complementary technique to the Infrared spectroscopy, in order to determine whether this method can also be used as a model for the screening of different embryo toxic substances. Preliminary results have been obtained in reflectance mode and show similar trend in results.

Metabolome, Raman spectroscopy, Infrared spectroscopy

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