CHAPTER 1 12 1 When identifying biochemical compounds in body fluids either using a targeted approach, which typically focuses on one or more related selected pathways of interest, or an untargeted approach, which aims to simultaneously measure a large number of metabolites, can be employed. The latter method is most commonly used in the field of metabolomics. Metabolomics is defined as the study of all low molecular weight compounds (<1500 Da) in a sample. Metabolites are the molecular endpoints of gene expression and cell activity and thereby represent, in a way, “the molecular phenotype of an organism”. The various types of -omics, i.e. genomics, epigenomics, transcriptomics, proteomic and metabolomic, relate to each other (Figure 1). Genomics is the study of the genome at the DNA level, as does epigenomics which investigates modifications of the genome expression. Transcriptomics investigates genomic expression at the RNA level, whereas proteomics interrogates proteins. Finally, metabolomics deals with the metabolome, so the complete set of small-molecule metabolites. Logically, changes in gene expression, enzymes and environmental factors can all have an effect on the “systems biology” and metabolite concentrations.73 The advantage of the untargeted/omics approach is that it allows for a rapid, concurrent identification and quantification of a multitude of metabolites in many samples at once. By measuring multiple metabolites, one gets a better understanding of the overall metabolomic networks involved. Important aspects and considerations of this method are the validation of the metabolites measured, validation of the used platform, as well as standardization of collection and storage methods.74, 75 Sample collection Although metabolomics is a proven, worthwhile approach for biomarker identification, sample collection needs to be done very meticulously as metabolically active cells in body fluids may alter the metabolomic profile ex vivo. It has been shown that inaccuracies in the pre-analytical steps cause low quality samples and even up to 80% of the laboratory measurement inaccuracies in daily clinical routine diagnostics.76-78 In the field of metabolomics, the stability of many metabolites and lipids is extremely variable, therefore, systematic or pre-analytical accidents and inconsistencies can have a great effect on compounds with a low stability and lead to high variability in the analytical data. Therefore, the most critical steps regarding the quality of one’s metabolomics data are related to the pre-analytical phase. Of note, each step should be well-considered, standardized and controlled to prevent degradation of sample quality and misinterpretation of findings during the analysis of data. Important issues to consider in metabolomics research are which biochemical fluid one intends to collect, whether the materials in the collection process up to sample preparation are suitable and do not interfere with the measurement method or the low-freezing storage facility.75 Another important step is to consider at what temperatures the body fluid will be kept during the preparation process, as lower temperatures reduce the activity of cellular metabolism. It is generally considered that it is important to centrifuge samples as soon as possible, but consistency in time until centrifugation is even more important.75 Therefore, a standardized protocol for the process of body fluids is essential. Other issues that are more obvious to be kept consistent are,
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