Procedure for tissue sample preparation and metabolite extraction for high-throughput targeted metabolomics

Abstract

Reproducible quantification of metabolites in tissue samples is of high importance for characterization of animal models and identification of metabolic changes that occur in different tissue types in specific diseases. However, the extraction of metabolites from tissue is often the most labor-intensive and error-prone step in metabolomics studies. Here, we report the development of a standardized high-throughput method for rapid and reproducible extraction of metabolites from multiple tissue samples from different organs of several species. The method involves a bead-based homogenizer in combination with a simple extraction protocol and is compatible with state-ofthe-art metabolomics kit technology for quantitative and targeted flow injection tandem mass spectrometry. We analyzed different extraction solvents for both reproducibility as well as suppression effects for a range of different animal tissue types including liver, kidney, muscle, brain, and fat tissue from mouse and bovine. In this study, we show that for most metabolites a simple methanolic extraction is best suited for reliable results. An additional extraction step with phosphate buffer can be used to improve the extraction yields for a few more polar metabolites. We provide a verified tissue extraction setup to be used with different indications. Our results demonstrate that this high-throughput procedure provides a basis for metabolomic assays with a wide spectrum of metabolites. The developed method can be used for tissue extraction setup for different indications like studies of metabolic syndrome, obesity, diabetes or cardiovascular disorders and nutrient transformation in livestock.

Identification of Serum Metabolites Associated With Incident Hypertension in the European Prospective Investigation Into Cancer and Nutrition–Potsdam Study

“…13 Analysis was done by the Genome Analysis Center at the Helmholtz Zentrum München. Please refer the study by Römisch-Margl et al 13 for analytic details.…”

“…13 Analysis was done by the Genome Analysis Center at the Helmholtz Zentrum München. Please refer the study by Römisch-Margl et al 13 for analytic details. Metabolites with concentrations below the detection limit or high analytic variance (n=36) were excluded, 14 leaving the following 127 quantified metabolites for statistical analyses (Table S1 in the onlineonly Data Supplement): hexose (sum of 6-carbon monosaccharides without distinction of isomers), 14 amino acids, 14 spingomyelins, 17 acylcarnitines (Cx:y; with x indicating carbon atoms and y indicating double bonds), and 81 glycerophospholipids (37 acyl-alkyl-, 34 diacyl-, and 10 lyso-phosphatidylcholines).…”

Alzheimer's disease–like pathology has transient effects on the brain and blood metabolome

“…Quantitative mass spectrometry-based metabolomic profiling was performed using the Biocrates AbsoluteIDQ p180 (BIOCRATES, Life Science AG, Innsbruck, Austria), as previously described (Nkuipou-Kenfack et al, 2014;Roemisch-Margl et al, 2012). The AbsoluteIDQ p180 kit provides simultaneous quantification of amino acids, acylcarnitines, SPHs, PCs, hexose (glucose), and biogenic amines in many biological samples.…”

Lactation is associated with altered metabolomic signatures in women with gestational diabetes

“…This assay can simultaneously quantify 188 metabolites in 10 μl plasma, including 40 acylcarnitines (Cx:y, where x indicates the total number of carbons and y the number of double bonds in the fatty acid side chains), 21 amino acids (19 proteinogenic plus citrulline and ornithine), 21 biogenic amines, total hexoses, 90 glycerophospholipids (14 lysophosphatidylcholines [lysoPCs] and 76 phosphatidylcholines [PCs]), and 15 sphingolipids. All analytical procedures were performed as previously described [21,22]. Briefly, 10 μl plasma was pipetted onto a filter which is incorporated into a 96-well sandwich plate and already contains internal standards stable isotope labelled with stable isotope.…”