For the cerebral cortex, this parameter is equal to 7.9%. However, the percentage of blood varies for different areas of the brain. The volume of blood in the brain of a mouse averages 3.5–4.0% of the total brain volume. In our work, we took the same sample volume (cerebral cortex tissue) from both groups of animals, so in the group of animals after the perfusion procedure, the entire volume of the sample contained only brain tissue, and in the group of animals in the native state, the sample consisted of brain tissue mixed with blood.
In the metabolomic analysis by in vitro NMR, the vessels are involved in the volume of the sample taken, and can potentially lead to a significant error. In this regard, a reasonable question arises about the potential contribution of biofluids to the errors in the analysis of the metabolism of tissue. Taking into account the structural and spatial organization of tissues may help us to avoid errors in the material intake, but separating all-penetrating biofluids from the tissues is often complicated. ĭeriving information on the metabolism of certain tissues may carry risks of sample contamination by other tissues and biological fluids of the organic body. Moreover, this method is suitable for identifying metabolites that can serve as markers of diseases such as cancer, diabetes, inborn metabolic disorders, and cardiovascular diseases. NMR data provide great interest in the development of approaches to the metabolic phenotyping of living organisms. Thus, it is possible to identify and determine the concentrations of many metabolites in a single run. The analysis of chemical shifts and multiplicities of NMR peaks allows us to reference them to specific metabolites. Moreover, a conventional NMR experiment does not require a complex procedure for sample preparation. The method of NMR spectroscopy is fast, non-invasive and provides highly reproducible results. One of the most powerful and versatile methods used in quantitative metabolomics studies is NMR spectroscopy.
In recent years, the number of applications of metabolomic tissue profiling for earlier diagnostics, monitoring the effectiveness of pathology therapy and assessments of the phenotype of living organisms has been rapidly increasing. (4) Thus, it was shown that the presence of blood does not have a significant effect on the metabolomic profile of the brain in animals without pathologies. (3) For the major set of studied metabolites, no significant differences were found in the brain tissue metabolite concentrations in the native state and after the blood removal procedure. We identified 36 metabolites in the brain tissue with the use of NMR spectroscopy. The group comparison was performed with the use of the Student’s test. Samples were studied by high-frequency 1H-NMR spectroscopy with subsequent statistical data analysis. The brain tissues of the animals were homogenized, and the metabolite fraction was extracted with a water/methanol/chloroform solution. The first group of animals ( n = 7) was subjected to the perfusion procedure, and the second group of animals ( n = 6) was not perfused. The animals were divided into two groups. We used 13 male laboratory CD-1 ® IGS mice for this study. (2) In this paper, the metabolomic profiling based on NMR spectroscopy was performed to determine the effect of blood contained in the studied samples of brain tissue on their metabolomic profile. An important factor, in this case, is the presence of blood in a tissue sample, which can potentially lead to a change in the concentration of tissue metabolites and, as a result, distortion of experimental data and their interpretation. (1) Recently, metabolic profiling of the tissue in the native state or extracts of its metabolites has become increasingly important in the field of metabolomics.